Immunoprecipitation strategies to isolate RIPK1/RIPK3 complexes in mouse macrophages

Abstract

A large protein complex, containing RIPK1, RIPK3 and caspase-8 and known as Complex II, has emerged as one of the key mediators of cell death downstream from a range of innate immune triggers. This regulatory mechanism plays a prominent role in macrophages where Complex II has been linked to apoptosis, pyroptosis and necroptosis as well as enhancement of the inflammatory gene expression. While core components of this complex are fairly well understood, more subtle proteomic changes that determine the direction of a response once the complex is assembled remain much less understood. In addition, Complex II components undergo a wealth of post-translational changes, that modify the functions of the complex components. This necessitates development of the robust and efficient methods for Complex II isolation for further interrogation of the complex composition and posttranslational modifications of its components. Our paper described several methods that we have developed for Complex II isolation, which can be used to obtain complimentary information about this signaling mechanism.

INTRODUCTION:

RIPK1 and RIPK3 kinases are key mediators of regulated necrosis mechanisms in macrophages. Activation of necrosis is mediated by a large cytosolic protein complex, termed Complex II. Both RIPK1 and RIPK3 undergo autophosphorylation in Complex II, and RIPK3 also directly phosphorylates the pseudokinase MLKL. The latter undergoes disulfide-bond reinforced oligomerization and serves as an indispensable executioner of necroptotic cell lysis. Depending on the cellular context, Complex II can also activate cysteine protease caspase-8, leading to apoptosis or the second form of regulated necrosis, pyroptosis. The latter occurs through processing, oligomerization and formation of lytic pores by Gasdermin D. The functions of various factors in Complex II are tightly controlled through a range of post-translational modifications and protein-protein interactions, thus making isolation of different versions of pro-death Complex II essential for the analysis of RIPK1 and RIPK3 signaling. Because Complex II is a large (up to megadalton MW) oligomeric complex, which segregates into the detergent insoluble compartment in the cells, its isolation requires modified conditions. In addition, heavily modified proteins in the complex tend to provide significant background that needs to be accounted for. We present the protocol for isolation of this Complex using an anti-FADD antibody (Basic protocol 1). Exogenous RIPK1 expression is difficult to achieve. We present a method for inducible expression of tagged RIPK1 and elution of RIPK1-containing complexes under mild conditions and discuss additional targets for immunoprecipitation (Basic protocol 2). Lastly, we present a proximity biotin labeling-based approach utilizing a TurboID tag to facilitate isolation of weakly or transiently bound components of the complex (Basic protocol 3).

BASIC PROTOCOL 1

Isolation of Complex II in necroptotic and pyroptotic macrophages using FADD immunoprecipitation.

Introductory paragraph:

Activation of cell death by RIPK1 and RIPK3 kinases requires assembly of a large cytosolic protein complex, termed, depending on context, Complex II, the necrosome or the ripoptosome. Complex II can provide the catalytic activities necessary for the execution of diverse types of cell death. In many cases, RIPK1 serves as a key initiator of complex formation, bringing in additional components such as RIPK3, which occurs through the oligomerization of the RIP Homotypic Interaction Motifs (RHIMs) (Li et al., 2012) and promotes RIPK3-mediated phosphorylation of MLKL, which is necessary for execution of necroptosis. Additionally, RIPK1 can recruit the adaptor protein FAS-associated protein with Death Domain (FADD) via homotypic interactions of the Death Domains (DDs). This, in turn, serves to recruit the cysteine protease caspase-8, responsible for the cleavage of executioner caspases or Gasdermin D (GSDMD) to drive apoptosis and pyroptosis, respectively (Micheau & Tschopp, 2003; Sarhan et al., 2018). Ultimately, the choice of the direction of the response is defined by the exact regulation in Complex II. For example, activation of caspase-8 is inhibitory for the induction of necroptosis, while the caspase-8 binding partner Cellular FLICE (FADD-like IL-1β-converting enzyme)-Inhibitory Protein Long (cFLIP_L) may operate as a switch between apoptosis and pyroptosis (Green, Oberst, Dillon, Weinlich, & Salvesen, 2011; Muendlein et al., 2020). Because FADD and caspase-8 recruitment occurs specifically into Complex II (Micheau & Tschopp, 2003), immunoprecipitation of these proteins has served as a reliable approach for Complex II isolation. In this section, we present a modified version of the FADD IP protocol, which allows efficient isolation of Complex II.

Materials:

• RAW 264.7 macrophages

• 1X Phosphate-Buffered Saline Diluted from 10X in dH₂O (Fisher, cat no. BP3994)

• DMEM/High Glucose (Corning, cat no. 10-017-CM)

• Lipopolysaccharides (LPS, from Escherichia coli 0111: B4; Sigma, cat no. L2630)

• IDN-6556/Emricasan (Medkoo Biosciences, cat no. 510230) or zVAD.fmk (SelleckChem, cat no. S7023)

• 5z-7-oxozeaenol (Cayman Chemical, cat no. 17459)

• Lysis Buffer (see recipe in Reagents and Solutions below)

• Protein Assay Reagent (Pierce 660nm, Thermo Scientific cat no. 22660, or equivalent)

• Mouse monoclonal anti-FADD antibody clone IF7 IgG (Millipore Sigma, cat no. 05-486)

• Normal anti-Mouse IgG (Santa Cruz Biotechnology, cat no. sc-2025)

• Protein A/G Beads (Thermo Scientific, cat no. 88802)

• Gelatin (Bio-Rad, cat no. 1706537)

• Ubiquitin Specific Protease 2 (USP2) (Boston Biochem, cat no. E-506-050)

• 1X SDS Loading Buffer Diluted from 4X in dH₂O (see recipe for 4X SDS loading buffer in Reagents and Solutions Below)

• Laminar Flow Hood (Labconco Purifier BSC Class II, or equivalent)

• Tissue culture microscope (Nikon or equivalent)

• 150mm Tissue Culture Treated Dishes (Fisher, cat no. FB012925)

• 15-mL Conical Tubes (Genesee scientific, cat no. 28-101)

• Plastic Sterile Cell scrapers

• 1.7mL Microcentrifuge Tubes (Olympus, cat no. 22-281)

• Fisher Vortex Genie 2

• Chilled Centrifuge Mikro 220R (Hettich Zentrifugen, or equivalent)

• 96-well Plate (Genesee Scientific, cat no. 25-109)

• VICTOR³V Multi Label Plate Reader (PerkinElmer, or equivalent)

• Bead Separation Magnetic Rack (Dynal Invitrogen, or equivalent)

• End-Over-End Rotator (Fisher)

• Heat block

Antibodies for Western Blot Analysis

• Anti-FADD mouse monoclonal clone IF7 IgG (Millipore Sigma, cat no. 05-486)

• Anti-RIPK1 rabbit monoclonal clone D94C12 (Cell Signaling, cat no. 3493)

• Anti-phospho-Ser166-RIPK1 rabbit monoclonal clone E7G6O (Cell Signaling, cat no. 53286)

• Anti-RIPK3 rabbit polyclonal (ProSci, cat no. 2283)

• Anti-phospho-Thr231/Ser232-RIPK3 rabbit monoclonal clone E7S1R (Cell Signaling, cat no. 91702)

• Anti-MLKL rat monoclonal clone 3H1 (Millipore, cat no. MABC604)

• Anti-phospho-S345-MLKL rabbit monoclonal clone EPR9515(2) (Abcam, cat no. ab196436)

• Anti-ZBP1 mouse monoclonal clone Zippy-1 (Adipogen, cat no. AG-20B-0010-C100)

• Anti-caspase-8 rabbit monoclonal clone D35G2 (Cell Signaling, cat no. 4790) or anti-cleaved-caspase-8(Asp387) rabbit monoclonal clone D5B2 (Cell Signaling, cat no. 8592)

• Anti-Tubulin rabbit polyclonal (Protein Tech, cat no. 11224-1-AP)

  Note: Because caspase-8, RIPK3, MLKL and FADD are similar in size to the heavy and light chains of IgG, additional measures may be needed to detect these proteins in the IPs. This may include crosslinking of the FADD antibody to the beads using cross-linking agents like BS3 and DMP (Pierce), selection of alternative primary antibodies from different species, use of conformation-selective secondary antibodies, such as Easyblot secondary antibodies (Genetex) or careful selection of acrylamide gel percentage in the SDS-PAGE step to maximally separate IgG bands. Please also note that use of crosslinking with FADD antibody in our tests resulted in a substantial but not complete loss of binding.

  Note: An example of FADD pulldown is shown in Figure 1A.

Figure 1. Isolation of Complex II in macrophages using FADD IP.

A) Primary BMDM macrophages were stimulated with 10 ng/mL LPS ± 100 nM, TAK1 inhibitor, 5z-7-oxozeaenol (5z7) for 30, 60 or 120 minutes to induce a mixture of caspase-8-mediated apoptosis and necroptosis. 50 μM zVAD or 10 μM Necrostatin-1 (Nec-1) were added to inhibit caspase and RIPK1 activity, respectively. Cells were lysed in Buffer A containing both 1% NP-40 and 0.5% Triton X-100, centrifuged at low speed (1000xg) and immunoprecipitated using anti-FADD antibody. Immunoprecipitated samples were analyzed by SDS-PAGE/western blot for the presence of Complex II components. B-E) RAW 264.7 macrophages were stimulated with 100 ng/mL LPS and 20 μM IDN6556 for 3 hrs to induce necroptosis. Cells were lysed in Buffer A containing both 1% NP-40 and 0.5% Triton X-100, Buffer B containing only 1% NP-40, or Buffer C containing only 0.5% Triton X-100 and immunoprecipitated using anti-FADD antibody (B). Following lysis in Lysis Buffer A, cells were centrifuged at either high speed (14,000x) or low speed (1000 × g) prior to immunoprecipitation (C). Protein A/G beads were blocked or left unblocked in 1% Gelatin prior to immunoprecipitation to reduce nonspecific binding to the beads (D). Immunoprecipitated samples were subjected to USP2 treatment with 400 ng of USP2 enzyme in DUB buffer prior to elution (E).

Reagents and Solutions:

Lysis Buffer A:

• 1% NP-40 (v/v)

• 0.5% Triton X-100 (v/v)

• 50 mM Tris-HCl, pH 7.2–7.4

• 150 mM NaCl

• 1 mM EDTA

• 1 mM EGTA

• 3 mM NaF

• 1 mM NaVO₃

• 10 mM β-glycerophosphate

• 1 μg/mL Aprotinin, Leupeptin and Pepstatin

• 50 μg/mL Phenylmethylsulfonyl Fluoride (PMSF) added to buffer directly before use

Lysis Buffer B:

• 1% NP-40

• 50 mM Tris-HCl, pH 7.2–7.4

• 150 mM NaCl

• 1 mM EDTA

• 1 mM EGTA

• 3 mM NaF

• 1 mM NaVO₃

• 10 mM β-glycerophosphate

• 1 μg/mL Aprotinin, Leupeptin and Pepstatin

• 50 μg/mL Phenylmethylsulfonyl Fluoride (PMSF) added to buffer directly before use

Lysis Buffer C:

• 0.5% Triton X-100

• 50 mM Tris-HCl, pH 7.2–7.4

• 150 mM NaCl

• 1 mM EDTA

• 1 mM EGTA

• 3 mM NaF

• 1 mM NaVO₃

• 10 mM β-glycerophosphate

• 1 μg/mL Aprotinin, Leupeptin and Pepstatin

• 50 μg/mL Phenylmethylsulfonyl Fluoride (PMSF) added to buffer directly before use

DUB Buffer:

• 50 mM NaCl

• 50 mM Tris-HCI, pH 8.0

• 5 mM DTT added directly before use

• Ubiquitin Specific Protease 2 (USP2, Boston Biochem, cat no. E506) added directly before use

  Note: Store at Room Temperature for up to 1 year

4X SDS Loading Buffer

• 200 mM Tris-HCl, pH 6.8

• 40% Glycerol (v/v) (Fisher, cat no. BP229)

• 8% (w/v) Sodium Dodecyl Sulfate (Fisher Scientific, cat no. BP166)

• 0.08 % (w/v) Bromophenol Blue (Fisher Scientific, cat no. BP114)

• 50 mM EDTA

• 4% β-mercaptoethanol (v/v) (Fisher Scientific, cat no. O43446I)

  Note: Store at room temperature up to 1 year

Protocol steps with step annotations:

Cell stimulation

• 1.1.Seed 5.0 × 10⁶ RAW 264.7 macrophage cells/plate in 150mm tissue-culture plates in 20 mL of complete DMEM 2 days prior to the start of experiment. Seed 2 plates per condition.
• 1.2.Stimulate cells in 10 mL of fresh complete DMEM with LPS (100 ng/mL) and IDN6556 (20 μM) for 3 hrs to induce RIPK1-dependent necroptosis. Maintain plates in an incubator at 37°C with 5% CO₂ for the duration of treatment.

  Note: Proper conditions for this experiment should include LPS+IDN6556 + anti-FADD Ab as well as Unstimulated + anti-FADD Ab, and LPS+IDN6556 + anti-Mouse IgG.

  Note: Other macrophage types such as primary BMDMs and immortalized BMDMs are treated in a similar manner to induce necroptosis. Because pyroptosis is not readily induced in RAW 264.7 cells in our tests, BMDM cells are recommended for activation of pyroptosis. For activation of a mixture of pyroptosis and apoptosis, cells are stimulated with LPS (10 ng/mL) and 100 nM of TAK1 inhibitor, 5z-7-oxozeaenol. Additionally, silencing of cFLIP_L promotes specific activation of pyroptosis in response to LPS alone (Muendlein et al., 2020).

Cell Lysis

• 1.3.Harvest both adherent and non-adherent cells in PBS. Collect non-adherent cells by collecting culture medium in a 15 mL conical tube. Wash each plate with 1.5 mL PBS and collect adherent cells by mechanically scraping into a microcentrifuge tube.
• 1.4.Pellet floating cells by centrifuging at 1,400x for 5 minutes and combine with adherent cells in the microcentrifuge tube. Re-pellet all combined cells by centrifuging at 5,000x for 3 minutes at 4°C.
• 1.5.Lyse cells by resuspending the cell pellet in 1mL of chilled Lysis Buffer. Incubate samples for 1 hr while rotating at 4°C, vortexing every 10 minutes at speed 4.5 for 10 seconds at a time to ensure complete lysis.

  Note: Complex II is composed of heavily modified proteins that form poorly detergent-soluble aggregates, which makes complex recovery more challenging. In this protocol we compared the use of 3 different lysis buffers. We found that Lysis Buffer A, containing both NP-40 and Triton X-100 detergents increases recovery of Complex II components as shown for RIPK1 (Fig. 1B), as well as reduces background from non-specific binding to the beads. Use of NP-40 alone (Lysis Buffer B) or Triton X-100 alone (Lysis Buffer C) attenuates detection.

• 1.6.After cell lysis is complete, centrifuge samples at 1000 × g, 4°C for 15 minutes to pellet non-lysed cells and nuclei.

  Note: Spinning at low speed will pellet cell debris and remove the nuclei from the lysate samples. We compared efficiency of spinning lysates at high-speed which is typically employed in IP protocols (14,000x) to low speed (1000 × g). As previously described (Moquin, McQuade, & Chan, 2013), high speed spinning pellets Complex II. Conversely, low speed spinning preserves Complex II for IP without increasing the background (Fig. 1C).

• 1.7.Collect the supernatant and normalize lysate concentrations across all samples using Pierce 660nm protein assay or similar.
• 1.8.Aliquot 60 μL of each lysate sample and dilute in 20μL 4x SDS buffer to be used as input for SDS PAGE/Western Blot analysis. Use the remaining lysate sample for immunoprecipitation.

Immunoprecipitation

• 1.9.Transfer 0.5–1mg of protein per sample in 0.5–1 ml of lysis buffer A into a chilled microcentrifuge tube. Add 2.5 μL (2.5 μg) of anti-FADD antibody per 1mg of protein to each sample except for the sample designated as the negative control. For the negative control sample, add equivalent volume (2.5 μg per 1 mg of protein) of anti-Mouse IgG.
• 1.10.Rotate samples overnight at 4°C.

  Note: Incubation of samples with the antibody for a minimum of 2 hours may be sufficient for immunoprecipitation and help preserve more labile components, but longer incubations may be more efficient. For the purposes of this experiment, samples were incubated with the antibody overnight.

Preparation of Protein A/G Beads

• 1.11.The following day, pipet the required amount of Protein A/G beads into microcentrifuges tubes. Recommended amount of beads is 15 μL of Protein A/G bead slurry per 1 mg of protein for each sample. Wash bead slurry by resuspending in 1 mL of chilled lysis buffer and placing the beads on the magnet to remove the supernatant. Thoroughly wash the beads by repeating the washing step 3X.

  Note: These washing steps remove the preservation buffer used for long-term storage of the A/G beads.

• 1.12.After the final wash, resuspend beads in 1 mL of 1% gelatin in lysis buffer and incubate for 1 hr at room temperature.

  Note: Incubating beads in 1% gelatin in lysis buffer has proven useful in decreasing the background of non-specific binding of proteins to beads, resulting in clearer protein bands visualized by western blot following immunoprecipitation (Fig. 1D). Gelatin can be difficult to fully resuspend. Prepare solution ahead of time to allow for thorough mixing. Stock can be stored at 4°C but needs to be melted at 37°C prior to use. Incubation of beads with gelatin should be done at room temperature as 1% gelatin forms a gel at 4°C.

• 1.13.Following incubation with gelatin, place samples on magnet and allow for beads to settle before removing all of gelatin solution. Wash 1X with Lysis Buffer A, transfer the cell supernatants (from 1.9) into tubes containing the washed protein A/G beads. Mix to resuspend the beads and rotate at 4°C for 2 hrs.

  Note: Extended incubations of protein complexes with primary Ab may lead to the loss of labile components. To minimize this, beads can be pre-incubated with primary Ab in 1% gelatin solution for 1 hr at RT with rotation. After a wash with lysis buffer, beads can be incubated with cell lysates for 2 hrs in 4°C with rotation.

USP2 Treatment

• 1.14.Following incubation, place the samples on the magnet and allow for beads to settle before carefully decanting the supernatant. Wash the beads by resuspending them in 1mL of lysis buffer and placing them back on the magnet. Repeat this process 3X to ensure thorough washing of the beads.

  Note: Prepare DUB buffer by aliquoting 300 μL into a clean microcentrifuge tube and add DTT to a final concentration of 5 mM and 2 μg of recombinant USP2 (400 ng per sample). DUB buffer can be prepared ahead of time, however USP2 and DTT should be added directly before use.

• 1.15.Resuspend the washed beads in 60 μL of DUB buffer and incubate at 37°C for 30 minutes to allow the reaction to proceed.

  Note: Proteins in complex II, such as RIPK1 and RIPK3, can be heavily polyubiquitinated, which can make signal detection in immunoprecipitation difficult. Use of USP2 improves detection as shown for RIPK1 (Fig. 1E).

• 1.16.Terminate the USP2 reaction by adding 20 μL of 4X SDS loading buffer and boil the samples at 95°C for 5 minutes.
• 1.17.Briefly centrifuge samples and place them on the magnet. Allow the beads to settle and carefully collect and transfer the supernatant containing the denatured proteins into a fresh tube. Samples can be stored at −20°C for future analysis by SDS PAGE/Western Blot.

  Please see further information in the Understanding Results section (below).

BASIC PROTOCOL 2

Isolation of the complexes formed by the conditionally expressed 3XFLAG-RIPK1 protein.

Introductory paragraph:

Core proteins involved in the execution of cell death, including RIPK1, RIPK3 and caspase-8 undergo multiple post-translational modifications that play key roles in the regulation of Complex II assembly, and selection of the form of cell death (Cockram et al., 2021; Delanghe, Dondelinger, & Bertrand, 2020; Oberst et al., 2010; Pop et al., 2011). These modifications, which include phosphorylations, ubiquitinations, proteolytic processing and others, are exerted through the interactions of the core Complex II components with various factors recruited into the Complex as well as those acting outside of the complex. An example of the latter regulation is the inhibitory phosphorylation of RIPK1 by MAPKAPK2 (MK2) kinase, which affects the cytosolic pool of RIPK1 and its ability to participate in Complex II assembly (Jaco et al., 2017). Thus, interactions both within and outside of Complex II can be informative. This requires direct analysis of the binding partners of key proteins involved in Complex II rather than Complex II isolation. This section describes methods to isolate RIPK1 complexes using inducible expression of 3XFLAG-RIPK1 protein and alternative approaches for immunoprecipitation of additional components involved in the regulation of Complex II. Because several key Complex II components (RIPK3, MLKL, caspase-8 and others) are similar in size to heavy chain of IgG, mild elution conditions using 3XFLAG peptide also facilitate complex analysis using Western blotting or mass spectroscopy.

Materials:

• RAW 264.7 macrophages

• pCW57-MCS1-P2A-MSC2(RFP) (Addgene, cat no. 80923) containing 3XFLAG-RIPK1 insert

• RAW 264.7 macrophages stably infected with pCW57-3XFLAG-RIPK1

• Doxycycline (Sigma, cat no. D9881-1G) dissolved in dH₂O

• Lysis Buffer (see recipe for Lysis Buffer A in Reagents and Solutions Basic Protocol 1)

• Pierce ^™ Anti-DYKDDDDK (FLAG) Magnetic Agarose beads (Thermo Scientific, cat no. A36797)

• 3XFLAG-Peptide Elution Buffer (see recipe in Reagents and Solutions Below)

• 4X SDS Loading Buffer (see recipe in Reagents and Solution Basic Protocol 1)

• 100mm Tissue Culture Treated Dishes (Genesee scientific, cat no. 25-202)

Additional Antibodies for Western Blot Analysis

• See Basic Protocol 1.

Reagents and Solutions:

3XFLAG Elution Peptide Stock Solution

• Resuspension buffer: 50 mM Tris, pH 7.4, 150 mM NaCl

• Dissolve 3XFLAG Peptide (Sigma, cat no. F4799) at a concentration of 25 μg/μL to create stock concentration

• Aliquot and store in −20°C for up to 1 year

  Note: Repeated freeze/thaw cycles are not recommended

• Prepare Elution Buffer by diluting 3XFLAG stock (1:100) to a final concentration of 250 ng/μL in Lysis Buffer right before use. Not recommended to store in Lysis buffer for future use.

Protocol steps with step annotations:

Cell stimulation

• 2.1.Seed 2.5 × 10⁶ RAW 264.7 macrophage cells/plate in 100mm tissue-culture plates in 10 mL of complete DMEM 2 days prior to the start of experiment. Seed 2 plates per condition.

  Note: Seed 2 plates per condition for a total of 4 conditions. Proper conditions for this experiment should include unstimulated RAW 264.7 macrophages, LPS+IDN6556 stimulated RAW 264.7 macrophages, unstimulated 3XFLAG-RIPK1-expressing RAW 264.7 macrophages, LPS+IDN6556 3XFLAG-RIPK1-expressing RAW 264.7 macrophages. Induce RIPK1 expression with 1 μg/mL of Doxycycline 12 hours prior to stimulation. Doxycycline should also be added to RAW 264.7 macrophages not expressing 3XFLAG-RIPK1 as a control.

• 2.2.On the day of the experiment, stimulate cells in 5mL of fresh media containing LPS (100 ng/mL), IDN6556 (20 μM), and Doxycycline (1 μg/mL) and incubate at 37°C, 5% CO₂ for 2 hours.

  Note: Timing of the experiment is critical. RIPK1 activation triggers its degradation, thus longer incubation may lead to lower recovery of RIPK1. In addition, 3XFLAG-RIPK1 cells undergo necroptosis faster than wild-type RAW 264.7 macrophages. Optimized time of harvest for RIPK1 expressing RAW cells was ~1.5–2 hrs post stimulation in our tests.

Cell Lysis

• 2.3.Harvest and lyse cells as described in Basic Protocol 1 (1.3–1.8).
• 2.4.Aliquot 45 μL of each lysate sample, mix with 15 μL of 4XSDS buffer and boil for 5 min at 95°C for SDS PAGE/Western Blot analysis. Use the remaining lysate sample for immunoprecipitation.

Preparation of Pierce ^™ Anti-DYKDDDDK (FLAG) Magnetic Agarose beads

• 2.5.Prepare beads for immunoprecipitation. Aliquot 25 μL of bead slurry per 1 mg of total protein lysate for each sample.

  Note: anti-FLAG antibody is supplied already pre-coupled to the magnetic agarose beads. All that is required for proper immunoprecipitation is washing of beads to remove preservation buffer and addition of anti-FLAG beads to the samples.

• 2.6.Wash the beads 2X by resuspending in 1 mL of lysis buffer and placing on the magnet to allow for the beads to settle before removing supernatant.

  Note: The washing step is essential to remove the preservation and buffer used for long-term storage of the anti-FLAG Magnetic Agarose beads.

• 2.7.After the last washing step, transfer the normalized lysates into washed anti-FLAG Magnetic Agarose beads and rotate at 4°C for 2 hrs.

  Note: Recommended incubation is 2 hours. Longer incubation time could result in higher signal but also greatly increased background.

3XFLAG Peptide Elution

• 2.8.Place samples on the magnet and allow for beads to settle and carefully discard the supernatant with a 1 mL pipette.
• 2.9.Wash the beads by resuspending the beads in 500 μL of lysis buffer. Place resuspended beads on the magnet, allow the beads to settle, and completely remove the supernatant. Repeat washing step 3X to ensure thorough washing of beads.
• 2.10.After the final wash step, fully remove all of the remaining lysis buffer from the beads and resuspend the beads in 45 μL of 3XFLAG Elution Buffer and incubate at room temperature for 30 minutes while rotating to elute proteins from the anti-FLAG magnetic beads.

  Note: Using the mild elution conditions of 3XFLAG Peptide elution allows for elution and analysis of RIPK1 interactors with minimal contamination from IgG. Elution with 1XSDS loading buffer results is high background from antibody leaching.

• 2.11.Place samples on the magnet and carefully collect eluates from the beads. Dilute eluates with 15 μL of 4X SDS loading buffer and incubate at 95°C for 5 minutes. Samples can be stored in −20°C until analysis by SDS PAGE/Western Blot (Fig. 2).

  Note: For samples that are to be analyzed by mass spectrometry, aliquot desired volume of sample for western blot analysis and store remaining elution fraction in −80°C until analysis. Additional tandem purification steps may be needed for mass spectrometry due to background remaining after FLAG IP.

Figure 2. Immunoprecipitation of RIPK1 containing complexes in 3XFLAG-RIPK1-expressing RAW 264.7 macrophages.

RAW 264.7 macrophages and 3XFLAG-RIPK1-expressing cells 100 ng/ml LPS, 20 μM IDN6556 for 2 hrs to induce necroptosis. All cells were treated with 1 μg/ml Doxycycline for 24 hr prior to the experiment. RIPK1 was immunoprecipitated using Anti-DYKDDDDK (FLAG) Magnetic Agarose beads. Cell lysates and immunoprecipitated samples were analyzed by SDS PAGE/Western Blot for the presence of Complex II components.

ALTERNATE PROTOCOL 1

Alternative methods of immunoprecipitation of RIPK1 and other Complex II related factors.

Introductory paragraph:

This section briefly describes alternative methods for the immunoprecipitation of endogenous Complex II components. The overall procedure for this protocol is identical to Basic Protocol 1, with the exception of the different antibodies that we propose to use. However, it is important to keep in mind that the proteins that we target in this protocol (RIPK1, TRAM, ZBP1) are involved in multiple pathways outside of Complex II, thus care should be taken to distinguish relevant vs. unrelated mechanisms.

Specifically, TRAM is a binding partner of the adaptor TRIF, a RHIM containing protein, which is required for necrosome formation in response to the TLR4 agonist LPS in macrophages (Kaiser et al., 2013; Najjar et al., 2016). TRAM is necessary for the endosomal trafficking of TRIF, where it forms secondary signaling complexes (Kagan et al., 2008), including Complex II. ZBP1 is another RHIM containing protein that plays many critical roles, including promoting formation of RIPK1-dependent Complex II under pyroptotic conditions and initiation of an alternative RIPK1 signaling pathway by engaging RIPK3 directly in response to Z-forms of nucleic acids upon viral infections (Muendlein et al., 2021; Zhang et al., 2020).

[Additional] Materials:

For RIPK1 immunoprecipitation, we found the combination of the rabbit monoclonal anti-RIPK1 antibody (Clone D94C12, cat no 3493, Cell Signaling) and mouse monoclonal anti-RIP1 antibody (Clone 7H10, cat no 72139, Abcam) provide optimal immunoprecipitation of RIPK1 from control and stimulated mouse and human cells. Dilutions of 1:100–1:500 are recommended for both antibodies.

For TRAM immunoprecipitation, use mouse monoclonal anti-TRAM antibody (Clone E-2, cat no sc-376076, Santa Cruz). Recommended amount is 1 μg per 0.25–1 mg of cell lysate.

For ZBP1 immunoprecipitation, use mouse monoclonal anti-ZBP1 antibody (clone Zippy-1, cat no AG-20B-0010-C100, Adipogen) at 1:250 dilution.

Reagents and Protocol are identical to Basic Protocol 1.

SUPPORT PROTOCOL 2

Support Protocols supplement the Basic or Alternate Protocol(s), e.g., to describe the preparation of a complex reagent or to describe steps that may be used in another context.

Generation of stable macrophage cell lines using lentiviral expression.

Introductory paragraph:

Primary and immortalized macrophages have relatively poor response to viral transduction, including limited efficiency of infection and tendency of the cells to undergo cell death. In addition, various proteins discussed in this manuscript can be harmful to the cells and are difficult to overexpress constitutively, especially RIPK1 and caspase-8. Generally, use of either LVX series of lentiviral vectors for constitutive expression or CW57-based all-in-one doxycycline inducible vectors has been successful in various macrophages using the optimized protocol described below.

Materials:

• pCW57-MCS1-P2A-MSC2 vectors, such as pCW57-MCS1-P2A-MSC2(RFP) (cat no. 80923, Addgene), which allows expression of two proteins using P2A self-cleaving peptide under the control of the Tet-inducible promoter. This vector also encodes a constitutively expressed cassette encoding TurboRFP, T2A self-cleaving peptide and rTetR protein.

• Lentiviral packaging plasmids, psPAX2 (Addgene, cat no. 12260) and pMD2.G (Addgene, cat no. 12259)

• HEK293T Cells

• Opti-MEM (Gibco, cat no. 11058-021)

• Lipofectamine 2000 (Invitrogen, cat no. 11668-027)

• 0.45μm PVDF Filter (Millipore, cat no. SLHVR33RS)

• 10mL Disposable Syringe without needle (Air-Tite cat no. ML10)

• Human Plasma Fibronectin (Millipore, cat no. FC010)

• T-75cm² Tissue Culture Flasks (Greiner Bio-One, cat no. 658170)

• 12-well non-Tissue Culture Treated plates (Falcon, cat no. 351143)

• 6-well Plate (Genesee Scientific, cat no. 25-105)

• Sorvall RT6000B Refrigerated Centrifuge, or equivalent

Reagent and Solutions

Virus Concentration Reagent

• 41% PEG 8000 (w/v) (Fisher Scientific, cat no. BP-233)

• 8.5% (w/v) NaCl (Fisher, cat no. S671)

• 20% (v/v) 1X PBS

• Autoclave and fully dissolve PEG while still hot and cool to room temperature

• Store at 4°C for up to 1 year

Protocol steps with step annotations:

Seed HEK293T Producer cells

• 1Seed 4.5–5.0 × 10⁶ cells in a T-75cm² Flask in 15mL of complete DMEM the day before transfection.
• 2On the day of the transfection, carefully decant media and replenish with 12mL of fresh DMEM without Antibiotic-Antimycotic 2–3 hours prior to transfection.

  Note: Use of media without Anti-Anti is only recommended for transfection. Following the day of transfection, cells should be replenished in complete DMEM.

Lipofectamine 2000 Transfection

• 3Prepare 2 microcentrifuge tubes with 600 μL of Opti-MEM designated “Tube 1” and “Tube 2”.
• 4Into “Tube 1” carefully add 48 μL of Lipo2000 Reagent and mix thoroughly. Into “Tube 2”, carefully add 7.2 μg of psPAX2 and 3 μg pMD2.G 3^rd generation lentiviral packaging plasmids and 9.6 μg of target vector pCW57-MCS1-P2A-MSC2-3XFLAG-RIPK1. Mix thoroughly and carefully transfer entire volume of “Tube 2” into “Tube 1” containing Lipo2000 Reagent and mix 1:1 by vortexing at speed 4.5.
• 5Incubate the DNA:Lipid mixture for 15 minutes at Room Temperature.
• 6Carefully add DNA:Lipid mixture to the 293T producer cells and ensure equal distribution across the entire flask by gently shaking the flask. Return the flask to the incubator at 37°C with 5% CO₂.

Virus Collection and Precipitation

• 716 hrs after transfection, remove the media and replenish with 10 mL of fresh media.
• 8After media change, collect virus supernatant twice, 24- and 48-hours post-media change and filter virus supernatant through a 0.45 μm PVDF filter. Store the filtered virus at 4°C.
• 9Precipitate the viral supernatant by diluting with virus concentration reagent (3:1) and incubate overnight at 4°C.

  Note: The virus concentration buffer is very viscous. Ensure proper mixing of contents for complete precipitation.

• 10Centrifuge viral precipitate at 1,500 × g for 45 minutes to pellet the virus.

RAW 264.7 macrophage Infection

• 11Macrophages can be difficult to infect. 24 hours prior to the day of infection, prepare a non-TC treated 12-well plate (Falcon, cat no. 351143) by coating with human plasma fibronectin (Millipore, cat no. FC010). To do this, add 50 μg of Fibronectin per well in 500 μL of PBS. The plasma fibronectin will serve to concentrate virions at the bottom of the wells, and upon addition of cells, the viral particles and cells will be in close proximity leading to higher efficiency of infection. We recommend the use of concentrated virus collected from one T75 flask per infection. Prior to the addition of the virus to the well, block fibronectin coated wells with 500 μL of 2% BSA in PBS.
• 12Resuspend pelleted virus from step (10) in 500μL of complete DMEM by repeated pipetting and carefully add the resuspended virus to a designated well in a non-TC treated 12-well plate pre-coated with Fibronectin. Bring final volume of each well to 1 mL with complete DMEM.
• 13Centrifuge plates containing viral particles at 2000 × g for 2 hrs to pellet the virus to the bottom of the well.
• 14Carefully remove supernatant and wash the wells with 1 mL of PBS to remove debris.
• 15Plate 1.5 × 10⁵ cells on top of the adhered virus.
• 16The following day, change the media in the infected wells and replenish with 1 mL fresh complete media.
• 1748 hrs following infection, transfer infected cells into a 6-well TC treated 6-wells and start selection.

  Note: If you are selecting using resistance markers, be sure to include a negative control of non-infected cells to determine when selection is complete. For the selection of fluorescent markers sort at least 5–10 × 10⁶ cells by flow cytometry.

BASIC PROTOCOL 3

Use of proximity labeling to identify necrosome components in the detergent insoluble fraction of the cell lysates.

Introductory paragraph:

Complex II is a very large (~2 MDa) protein complex containing oligomerized core components, such as RIPK1, RIPK3, FADD and caspase-8 (Tenev et al., 2011). Other components are likely present in much lower quantities or interact transiently. Thus, the identification of new Complex II components may benefit from using additional approaches that allow capture of transient interactors, such as proximity labeling using a TurboID biotin ligase cassette (Branon et al., 2018). In this section, we described application of the TurboID approach to isolate necrosome-associated factors using TurboID-RIPK3 as bait.

Materials:

• RAW 264.7 macrophages

• pLVX-EF1a-IRES-zsGreen1 (Takara, cat no. 631982) vector containing TurboID-mRIPK3 insert

• RAW 264.7 macrophages stably infected with pLVX-TurboID-RIPK3

• SM-164 (MedChemExpress, cat no. HY-15989

• Biotin (Sigma, cat no. B4501)

• Necrosome Lysis Buffer (see recipe in Reagents and Solutions Below)

• Pierce Streptavidin Magnetic Beads (Pierce, cat no. 88816)

• 1XSDS w/out Bromophenol Blue (see recipe in Reagents and Solutions Below)

• Thesit Buffer (see recipe in Reagents and Solutions Below)

• Tris-Buffer Saline with 1% Tween (v/v), Tween 20 (Amresco cat no. 500-018-3)

• Wash Buffer A (0.1% SDS, 1% NP-40, in PBS)

• Wash Buffer B (0.1% SDS, 1% NP-40, 400 mM NaCl, in PBS)

• 50 mM Tris-HCl, pH 7.4

• Elution Buffer (2% SDS, 4 mM Biotin in PBS)

• 4XSDS Loading Buffer (see recipe in Reagents and Solutions Basic Protocol 1)

Additional Antibodies for Western Blot Analysis

• Pierce High Sensitivity Streptavidin-HRP (Pierce, cat no. 21130)

Reagents and Solutions:

Necrosome Lysis Buffer:

• 0.1% Triton X-100 (v/v)

• 150 mM NaCl

• 1 mM EDTA

• 20 mM Tris-HCl, pH 7.5

• 3 mM NaF

• 1 mM β-glycerophosphate

• 1 mM NaVO₃

• 1 μg/mL Aprotinin, Leupeptin, Pepstatin

• 50 μg/mL Phenylmethylsulfonyl Fluoride (PMSF) added to buffer directly before use

• Prepare day of use, storing longer than overnight at 4°C not recommended.

1XSDS Buffer without Bromophenol Blue:

• 4X SDS Sample buffer containing no bromophenol blue diluted 1:3 with dH₂O

• Can be stored at Room Temperature up to 1 year

  Note: Buffer can solidify at lower temperatures. You can bring the buffer back into solution by heating up to 37°C. Vortexing during heating to 37°C can help facilitate this process.

Thesit Buffer:

• 2.5% Thesit (Sigma, cat no. 88315)

  Note: Thesit needs to be melted at 37°C before use.

• 50 mM HEPES, pH 7.5

• 150 mM NaCl, pH 7.5

• Can be stored at Room Temperature up to 1 year

Protocol steps with step annotations:

Cell Stimulation

• 3.1.Seed 2.5 × 10⁶ cells/plate in 100mm tissue-culture treated plates as described in Basic Protocol 2.

  Note: Seed 2 plates per condition. Appropriate conditions for this experiment are unstimulated TurboID-RIPK3 cells, LPS+IDN6556 stimulated TurboID-RIPK3 cells, and RAW 264.7 macrophages stimulated with LPS+IDN6556, as a negative control.

• 3.2.Treat cells with 5 mL of fresh complete DMEM with LPS (100 ng/mL) and IDN6556 (20 μM), SM164 (1 μM), Biotin (500 μM) for 1.5 hrs to induce necroptosis. Maintain plates in an incubator at 37°C with 5% CO₂ for the duration of treatment.

  Note: Control plates should be treated with an equal volume of DMSO. Cells expressing mRIPK3-TurboID die faster than WT cells. Optimal time to harvest RIPK3 expressing RAW cells is 1.5 hrs post stimulation.

Cell Lysis & Sample Preparation

• 3.3.Harvest cells as described in Basic Protocol 1 (1.3–1.4) and resuspend in 500 μL of Necrosome Lysis Buffer.
• 3.4.Lyse resuspended cells by placing them on ice for 15 minutes and vortex samples 3–4 times at low speed (setting 4.5) for 10 seconds at a time to ensure mixing.
• 3.5.Centrifuge lysed samples at 1000 × g at 4°C for 15 minutes to pellet nuclei.

  Note: Spinning at low speed will pellet cell debris and remove the nuclei from the lysate samples. Spinning at higher speeds will pellet necrosomes and is not recommended.

• 3.6.Collect supernatant and normalize lysate concentrations across all samples as described in Basic Protocol 1 (1.7) and centrifuge the normalized lysate samples at 14,000x for 15 minutes at 4°C to pellet necrosomes. Collect the supernatant and wash the pellet with an equal volume of lysis buffer (500 μL) and repeat the spin to precipitate the Triton-insoluble necrosome pellet.

  Note: The supernatant collected after the first 14,000x spin represents the detergent-soluble lysate fraction. Dilute eluates with 15 μL of 4XSDS loading buffer and incubate at 95°C for 5 minutes. Use the remaining soluble lysate sample for immunoprecipitation.

• 3.7.After the second 14,000x spin, completely remove the supernatant, resuspend the Triton-insoluble pellet in 100 μL of 1XSDS buffer (without bromophenol blue), and boil samples for 5 minutes at 85–90°C.

  Note: Boiling the pellet in 1XSDS buffer will solubilize the detergent insoluble material. However, in order to proceed with the pulldown SDS needs to be neutralized with Thesit buffer.

• 3.8.Neutralize SDS by adding 4 volumes of Thesit buffer to each sample (400 μL). Aliquot 45 μL of the insoluble lysate fraction and dilute in 4XSDS buffer for SDS PAGE/Western Blot analysis. Use the remaining necrosome-enriched solubilized sample for immunoprecipitation.

Bead Preparation & Immunoprecipitation

• 3.9.Prepare Pierce Streptavidin Magnetic beads for immunoprecipitation. Pre-wash the beads with 500 μL of TBS with 0.1% Tween by fully resuspending beads and placing beads on the magnet. Repeat this wash step twice and transfer the normalized soluble and insoluble lysate fractions into the washed beads and mix to fully resuspend.

  Note: Beads should be added to samples based on the protein concentration. The recommended amount is 15 μL of bead slurry for 400 μL lysate sample at a protein concentration of 1 mg/mL. Adding excess beads can increase the background.

• 3.10.Rotate samples with beads at 4°C for 2 hours.
• 3.11.Place tubes containing magnetic bead/protein slurry on the magnet and let beads settle before discarding the flow-through. Wash the beads by resuspending them and placing them back on the magnet. Wash 3X with 500 μL of Wash Buffer A, 2X with 500 μL Wash Buffer B, and 1X with 500 μL 50mM Tris-HCl pH 7.4.

  Note: After each wash, allow beads to fully settle before discarding supernatant. Not allowing beads to settle can result in loss of beads and sample.

• 3.12.Elute biotinylated proteins from streptavidin beads by fully resuspending beads in 45 μL of Elution Buffer. Incubate the resuspended beads at room temperature for 15 minutes, followed by boiling for an additional 15 minutes at 85–90°C. Once boiling is finished, place the beads back on the magnet and carefully collect elution from the beads and discard the remaining beads.
• 3.13.Prepare samples for western blot analysis by adding 15 μL of 4X SDS and boil at 95°C for 5 minutes. Store samples in −20°C until analysis by SDS PAGE/Western Blot (Fig. 3).

  Note: Endogenous mRIPK3 is approximately 56 kDa. TurboID-mRIPK3 produces a band of approximately 85–90 kDa. For samples that are to be analyzed by mass spectrometry, aliquot desired volume of sample for western blot analysis and store remaining elution fraction in −80°C until analysis.

Figure 3. Isolation of biotinylated proteins from Triton-soluble and insoluble fractions in TurboID-RIPK3-expressing RAW264.7 macrophages proximity.

RAW 264.7 macrophages and TubroID-RIPK3-expressing cells were stimulated with 100 ng/ml LPS, 20 μM IDN6556, and 100 nM SM164 (LIS) for 1.5 hrs to induce necroptosis. Cells were lysed in Necrosome lysis buffer and separated into Triton-soluble vs insoluble fractions before immunoprecipitation using Streptavidin Magnetic beads to isolate biotinylated proteins. Samples were run on SDS-PAGE/Western blot and analyzed for the presence of Biotin and RIPK1.

COMMENTARY:

Background Information:

The complex of RIPK1, RIPK3 and caspase-8 has emerged as a key activator of diverse cell death mechanisms in multiple cell types, including macrophages. The mechanism of signaling by Complex II is described in multiple recent reviews, including ours (Degterev, Ofengeim, & Yuan, 2019). Formation of this critical protein complex is best characterized in the case of signaling downstream from TNF receptor 1 (TNFR1). Binding of TNFα to TNFR1 leads to the rapid formation of receptor bound Complex I (Micheau & Tschopp, 2003) including TRADD, RIPK1 and a variety of regulatory factors (cIAP1/2, LUBAC complex, others) which serve to nucleate the assembly of an NF-kB activating complex including kinases TAK1 and the trimeric IKKα/β/γ kinase complex. Complex I assembles on poly-ubiquitinated RIPK1 which, in this case, acts as a scaffold rather than a kinase. An additional critical function of the E3 ligases and kinases associated with Complex I is to prevent formation of the secondary cell death-inducing Complex II. Complex II results from the dissociation of RIPK1 and TRADD from the receptor and recruitment of FADD/caspase-8 and RIPK3. While one of the versions of Complex II (sometimes termed Complex IIa and initially described by Micheau and Tschopp (Micheau & Tschopp, 2003)), leads to activation of caspase-8 and apoptosis in a RIPK1-independent manner, under many circumstances variants of Complex II dependent on RIPK1 and its catalytic activity are formed and activate apoptosis, necroptosis and pyroptosis. Choice of the cell death mechanisms is a prerogative of the different catalytic activities in the complex. High levels of caspase activity lead to apoptosis and pyroptosis. The latter may predominate in macrophages if the cells lack expression of the inactive homolog and caspase-8 binding partner cFLIP_L (Muendlein et al., 2020). Pyroptosis is mediated through caspase-8-dependent cleavage of Gasdermin D (GSDMD), which forms lytic pores in the plasma membrane (Sarhan et al., 2018). Conversely, lack of caspase-8 activity leads to activation of necroptosis, which proceeds through oligomerization and activation of RIPK3, which in turn phosphorylates pseudo-kinase MLKL. The latter undergoes a conformational change, forms disulfide-enforced oligomers which insert into plasma membrane and cause cell lysis (Frank & Vince, 2019).

Toll-like receptors TLR3 and TLR4 efficiently activate RIPK-dependent cell death pathways through the secondary endosomal complex involving adaptor TRIF (Kaiser et al., 2013; Najjar et al., 2016; Schworer et al., 2014). In contrast, the TLR4-bound MyD88 complex does not appear to play a major role in this regulation. Generally, the dynamics of Complex II assembly and regulation in this case are not yet completely understood. For example, it is not well established whether an analog of Complex I forms in TRIF pathway, and at what level(s) some of the inhibitory events observed in TNFR1 Complex I, such as RIPK1 ubiquitination and phosphorylation by cIAP1/2, LUBAC, TAK1 and the IKK complex, occur in the TRIF-mediated signaling pathway. However, it is clear that TLR signaling also involves formation of a large, detergent insoluble Complex II responsible for the initiation of cell death (Muendlein et al., 2021; Muendlein et al., 2020; Najjar et al., 2016; Saleh et al., 2017). In addition to regulation of cell death, RIPK1 and RIPK3 in Complex II also promote inflammatory gene transcription and translation, contributing along with the Danger Associated Molecular Patterns (DAMPs) released by lysed cells to the inflammatory nature of TLR-induced cell death. Because Complex II plays a central role in the regulation of multiple cell death mechanisms, efficient proteomic approaches to characterize Complex II are paramount.

Critical Parameters:

General considerations

Regulation of RIPK dependent cell death is subject to a large number of general and cell type specific controls, which need to be taken into account in the experimental design. For example, up to 80% of widely used cancer cell line’s lack the ability to activate necroptosis in response to TNFα (Najafov et al., 2018). Similarly, activation of GSDMD-mediated pyroptosis is generally restricted to myeloid cells. The timing of the analysis also plays a critical role. Initial receptor-bound complexes assemble on TNFR1 and TLRs in a matter of minutes. However, the secondary death-inducing Complex II typically assembles with significantly delayed kinetics (>2 hrs). Thus, establishing the kinetics of cell death pathway activation, which can be readily assessed using phospho-specific antibodies against RIPK1, RIPK3 and MLKL and antibodies detecting processed fragments of caspases and GSDMD, is essential before starting Complex II isolation experiments. In addition, neither TNFα nor TLR agonists alone induce cell death in the majority of cell types, including macrophages. Co-treatment with TAK1 inhibitor 5z-7-oxozeanol, SMAC mimetics and caspase inhibitors are needed to overcome upstream inhibition of cell death. Thus, specific conditions allowing Complex II assembly in a particular model system may need to be identified.

Cell lysate preparation

Complex II is a poorly detergent soluble oligomeric complex and choice of proper cell lysis conditions is critical for the efficient isolation. In our tests, a combination of both Triton X-100 and NP-40 in the lysis buffer greatly increased complex recovery compared to the use of either detergent individually. In addition, a more active and extended lysis is beneficial, i.e., rotation for 1 hr in the cold room. At the same time, higher detergent concentrations and more vigorous disruption conditions, e.g. sonication, may reduce complex recovery and lead to the loss of the more labile components. In addition, clearing of the lysates using typical centrifugation at the maximal speed of the microcentrifuge is detrimental as it pellets the majority of the Complex II (Moquin et al., 2013). Thus, a slower spin at 1,000 × g is highly recommended to pellet nuclei and un-lysed cells. More crude lysates however could result in higher background necessitating more thorough washes.

Protein A/G bead preparation

Heavily modified RIPK1 and RIPK3 have a tendency to non-specifically accumulate on the beads. Increasing hydrophilicity of the beads along with blocking non-specific binding sites using preincubation with 1% gelatin is beneficial. It is also recommended to reduce bead incubation times for the samples. If high background is observed and also to reduce the dissociation of more labile components, antibody can be pre-bound to the beads for 1 hr at room temperature prior to the addition to cell lysates and incubation time can be reduced to 2 hrs at 4°C.

Use of USP2 DUB

RIPK1 and RIPK3 are heavily ubiquitinated in Complex II. Thus, to more accurately estimate the amounts of these proteins in the pulldowns it is recommended to include the bead treatment with the USP2 enzyme.

Enrichment of Complex II components in the detergent insoluble fraction

In our tests, the TurboID reaction resulted in limited selectivity between control and stimulated conditions. Thus, to ensure more selective isolation of necrosome-associated proteins, insoluble complexes pelleted by 14,000x spin were collected, solubilized and used for the isolation of the biotinylated proteins. By our estimates, that led to some but not complete enrichment of the necrosome components in the IPs. Duration of biotin addition could potentially be used as another parameter to control amount of labeling. However, cells may contain sufficient levels of biotin, which may render TurboID reaction independent of the exogenously added biotin. It is recommended that the effect of exogenous biotin addition on the signal is determined and used to select timing of biotin addition after the initiation of cell death.

Troubleshooting:

Table I.

Troubleshooting Guide for Isolation of Complex II in necroptotic and pyroptotic macrophages using FADD immunoprecipitation.

Problem	Possible Cause	Solution
Poor signal	Cells may not have responded to the treatment and thus did not undergo necroptosis or pyroptosis	Check the phenotype of treated cells before harvest. RAW 264.7 macrophages are very sensitive to necroptosis. Necrotic cells will quickly detach from the plate and need to be collected from the media. Optimization of cell death inducing conditions may be needed. Amounts of cells can be increased. Induction of cell death for an extended period of time may result in degradation of Complex II components.
High Background	Improper washing of the beads/removal of contaminants	If you are getting a lot of background signal, it may require that you wash the beads more thoroughly to remove contaminants from the beads. Ensure complete removal of buffer between each wash step and elution. Increasing duration of the washes (such as rotation for 30 min at 4 °C) and increasing salt and detergent concentrations may help reduce the background. Additionally, limiting protein concentrations in the lysate may be helpful.
Signal is too Strong	Using lysate at too high concentration for immunoprecipitation.	Using too high of a protein concentration can result in background and saturation of FADD antibody binding. Recommended amount of protein to be used for immunoprecipitation is 0.5–1mg.

Table II.

Troubleshooting Guide for identifying necrosome components in the detergent insoluble fraction of the cell lysates.

Problem	Possible Cause	Solution
Poor signal	Using too few cells results in low protein concentration making it difficult to detect biotinylated proteins in the insoluble fraction.	We recommend you use at least 2 dense 10 cm2 plates per condition (~80% confluency on the day of the experiment). This should yield sufficient protein (~1–2 mg) to allow for the detection of biotinylated proteins in the insoluble fraction.
Over-exposed western blot with Streptavidin-HRP	Extensive washing required	Using Streptavidin-HRP can result in very dark and over-exposed western blots. Finding the required dilution, exposure and washing conditions can be difficult. Significantly reducing Streptavidin-HRP concentration and extending the duration of washes or incubating wash overnight at room temperature can help reduce the background.

Understanding Results:

Isolation of Complex II using FADD Immunoprecipitation

Using anti-FADD immunoprecipitation is an effective way to isolate components of Complex II. Checking for the presence of RIPK1 and ph-Ser166-RIPK1 is an easy way to ensure efficiency and success of complex II isolation (Fig. 1). Identification of some Complex II components could be more difficult due to the overlap with IgG bands.

Isolation of the complexes formed by the conditionally expressed 3XFLAG-RIPK1 protein

Immunoprecipitation of Complex II using 3XFLAG-RIPK1 protein provides the opportunity for mild elution conditions without IgG, facilitating detection of necrosome components such as RIPK3, caspase-8, and FADD (Fig. 2). While there is limited background with anti-FLAG beads, we found the purity after a single step purification was generally not sufficient for mass spectrometry analysis, necessitating tandem approaches. Use of cells expressing 3XFLAG-RIPK1 with tagged RIPK3, FADD or catalytically inactive caspase-8 may facilitate complex isolation using tandem double IP. It should also be taken into account that many additional interactions with RIPK1, unrelated to Complex II, may be observed. It should be noted that the exogenous expression of RIPK1 that we were able to achieve is on par with the endogenous protein levels, which is likely due to the toxicity of overexpressed RIPK1.

Proximity Labeling of Necrosome Components

Combining the isolation of the detergent insoluble proteins with a labeling system such as TurboID-RIPK3 allows enrichment of the labeled necrosome components. However, because TurboID-RIPK3 itself accumulates in the insoluble fraction, the real enrichment of the specific RIPK3 targets is difficult to assess. In our tests, this approach yielded necrosome-enriched components based on mass spectrometry analysis. However, we recommend analyzing both soluble and insoluble fractions and including multiple negative controls, such as cells lacking expression of TurboID, using media without biotin, and determining if the same proteins are enriched in both soluble and insoluble fractions to maximize identification of specific vs. non-specific interactors (Fig. 3).

Time Considerations:

Seeding cells and allowing them to grow to appropriate density for the experiment requires approximately 2 days.

Rate at which cells undergo necroptosis or pyroptosis can vary between cell types. RAW 264.7 macrophages are very sensitive to necroptosis, and exogenous expression of RIPK1 or RIPK3 will further speed up the rate with which they die. 1.5–4 hrs of stimulation is sufficient for induction of cell death. Close attention should be paid to specific protocols noted above.

Cell lysis in Basic Protocols 1, 2, 3 takes approximately 1 hour to ensure proper cell lysis.

Immunoprecipitation times vary depending on antibody being used. For immunoprecipitation using anti-FADD antibody described in Basic Protocol 1 longer incubation times have proven useful. However, immunoprecipitation using anti-TRAM, anti-FLAG or anti-Streptavidin antibody require shorter incubation times of 2–3 hrs.

Elution of proteins in Basic Protocols 1, 2, or 3 take approximately 1 hr to complete. In each outlined protocol, elution step includes a short incubation and treatment to elute and denature proteins for SDS-PAGE/western blot.