Protocol for detection of cGAMP-induced STING oligomerization in cultured cells

Summary

STING (stimulator of interferon genes) plays a critical role in the innate immune response, including in viral infection, cancer immunity, and several autoimmune diseases. Here, we present a protocol for detection of STING oligomerization, a critical step in STING activation. We describe steps for expressing STING in HEK293T cells, activating STING by 2′3′ cyclic GMP-AMP (cGAMP) treatment, and analyzing STING oligomerization by non-reducing SDS-PAGE and blue native PAGE. This protocol is useful for studies of STING functions, regulation, and dysregulation.

For complete details on the use and execution of this protocol, please refer to Liu and Manley.¹

Graphical abstract

Highlights

• •Procedures for expressing and activating STING in HEK293T cells
• •Instructions for non-reducing SDS-PAGE and blue native PAGE
• •Steps for the analysis of STING oligomerization

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

STING (stimulator of interferon genes) plays a critical role in the innate immune response, including in viral infection, cancer immunity, and several autoimmune diseases. Here, we present a protocol for detection of STING oligomerization, a critical step in STING activation. We describe steps for expressing STING in HEK293T cells, activating STING by 2′3′ cyclic GMP-AMP (cGAMP) treatment, and analyzing STING oligomerization by non-reducing SDS-PAGE and blue native PAGE. This protocol is useful for studies of STING functions, regulation, and dysregulation.

Before you begin

Upon detection of cytosolic double-stranded DNA, cyclic GMP-AMP synthetase (cGAS) produces the second messenger cGAMP,² which binds to and activates the endoplasmic reticulum transmembrane protein STING^3,4 (Figure 1A). Activated STING triggers the signaling cascades of the IRF3, NF-κB and autophagy pathways, leading to production of immune cytokines and autophagosomes.^5,6,7,8 This process, known as the cGAS-STING pathway, plays a critical role in viral infection, cancer immunity and several autoimmune diseases.^9,10

Figure 1.

Oligomerization is a critical step in STING activation

(A) Schematic of the cGAS-STING pathway. The double-stranded DNA sensor cGAS produces cGAMP, which binds to and activates STING, leading to STING oligomerization. dsDNA: double-stranded DNA.

(B) Workflow of detection of STING activation via oligomerization. First express STING in cells, then activate STING by cGAMP. Finally detect STING oligomerization by either non-reducing SDS-PAGE or BN-PAGE.

STING oligomerization following cGAMP binding is an important step in STING activation¹¹ (Figure 1A). Below we describe a protocol for detection of STING oligomerization in HEK293T cells. HEK293T cells are ideal for this purpose because they do not express endogenous cGAS or STING¹² and are highly transfectable, thus allowing functional studies of various STING mutants and expression or manipulation of putative STING regulators. This protocol entails three major steps (Figure 1B). First, we transfect HEK293T cells with STING plasmid. We then activate the transfected STING with cGAMP. Finally, we detect STING oligomerization by non-reducing SDS-PAGE and blue native PAGE (BN-PAGE). Non-reducing SDS-PAGE is for detection of disulfide bond-linked STING oligomers, while BN-PAGE, which uses the dye Coomassie brilliant blue G250 (hence “blue”) to add a net negative charge to proteins,¹³ is for detection of STING oligomers both with and without disulfide bond linkage. This protocol is likely applicable to other cell lines, but readers should perform optimization for the specific cell lines being studied. Using this protocol, we recently successfully demonstrated that a non-canonical protein isoform of the mRNA polyadenylation factor WDR33, produced by exonic/intronic alternative polyadenylation,¹⁴ is an inhibitor of STING oligomerization.¹

CRITICAL: Always handle HEK293T cells inside a certified tissue culture hood. Sterilize all materials described below before bringing them into the hood. Always follow safety guidelines for all steps described in the protocol. Discard waste materials properly according to local regulations.

Preparation of HEK293T cells

Timing: 6–7 days

Note: Skip this section if there is an existing HEK293T cell culture that has not been passaged for more than 20 times after thaw.

• 1.Sterilize the work area of a certified tissue culture hood and all equipment/materials for tissue culture with 70% ethanol.
• 2.Warm up DMEM supplemented with 10% fetal bovine serum (FBS).

Note: Warm up at 25°C–37°C. “DMEM” hereafter refers to DMEM supplemented with 10% FBS.

CRITICAL: Lipofectamine 2000 manufacturer (Invitrogen) does not recommend performing transfection in the presence of antibiotics, such as penicillin and streptomycin. Make sure that DMEM used to culture cells for transfection does not contain any antibiotics.

• 3.Quickly thaw a frozen vial of HEK293T cells in a clean 37°C water bath.
• 4.Transfer the cell suspension from the vial to a 15 mL tube containing 4 mL DMEM.
• 5.Centrifuge the cell suspension at 300 x g for 5 min at 20°C–25°C.
• 6.Aspirate the supernatant.
• 7.Resuspend the cell pellet in 5 mL DMEM.
• 8.Transfer all resuspended cells dropwise to a 10-cm tissue culture dish containing 5 mL DMEM. Rock the dish to evenly distribute the cells.
• 9.Incubate the cells in a humidified 37°C CO₂ tissue culture incubator.
• 10.Perform the following steps to passage the cells when they become confluent.

Note: Check cell confluency daily. In our lab it typically takes 2–3 days after thaw for HEK293T cells to become confluent. Use cells that have been passaged at least once for experiments.

• 11.Warm up DMEM and 0.05% Trypsin-EDTA.
• 12.Aspirate the medium from the 10-cm dish containing confluent HEK293T cells.
• 13.Gently add 5 mL PBS to different area of the dish dropwise. Rock the dish to evenly distribute PBS.

Note: HEK293T cells detach easily from tissue culture dishes, but it is okay to lose a small fraction of cells here.

• 14.Aspirate the PBS. Add 1 mL trypsin-EDTA. Rock the dish to evenly distribute trypsin-EDTA.
• 15.Incubate the dish at 20°C–25°C. Rock and tap the dish occasionally until all cells are detached (1–3 min).
• 16.Add 9 mL DMEM. Pipette the cell suspension up and down until single cells are obtained.
• 17.Transfer 0.5 mL cell suspension to a new 10-cm dish containing 9.5 mL DMEM. Rock the dish to evenly distribute the cells.
• 18.Incubate the cells in a humidified 37°C CO₂ tissue culture incubator.

Note: These cells will be ready for experiments when they become confluent in ∼3 days.

Preparation of SDS-PAGE gel

Timing: ∼45 min

Note: Skip this section if you use precast gels.

• 19.Clean 0.75 mm spacer and top plates for PAGE gel with 70% ethanol.
• 20.Assemble the gel casting apparatus.

Note: Different gel casting apparatuses have different designs. We use Mini-PROTEAN (Bio-Rad). You may follow the manufacturer's instructions.

• 21.Add 3.5 mL 10% SDS separation gel solution to the space between the top and the spacer plates.

CRITICAL: Add the solution slowly and avoid making bubbles. You may tap the plates a few times to evenly distribute the solution and to remove small bubbles.

• 22.Immediately add 500 μL isopropanol to the top of the separation gel.

CRITICAL: Do not mix the isopropanol with the SDS separation gel solution.

• 23.Let the SDS separation gel solidify at 20°C–25°C for 15–20 min. Troubleshooting 1.
• 24.Decant the isopropanol. Wash the top of the separation gel with distilled water.
• 25.Decant the distilled water. Add sufficient SDS stacking gel solution to completely fill the space between the top and spacer plates (usually 1 mL).
• 26.Insert a 0.75 mm comb to the space between the top and spacer plates.

Note: Wipe out the overflowed SDS stacking gel solution.

• 27.Let the SDS stacking gel solidify at 20°C–25°C for 10–15 min.
• 28.Use the gel immediately or wrap it in wet paper towels with saran wrap and store at 4°C for no more than 3 days.

CRITICAL: Do not extract the gel out. Keep the gel inside the glass plates until electrophoresis is completed.

Preparation of BN-PAGE gel

Timing: ∼45 min

• 29.Repeat Steps 19-28 above but use 10% native separation and native stacking gel solutions instead of 10% SDS separation and SDS stacking gel solutions, respectively. Troubleshooting 1.

CRITICAL: BN-PAGE gel must not contain SDS.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse anti-HA, 1:10,000	BioLegend	Cat# 901533; RRID: AB_2801249
Rabbit anti-mouse IgG, HRP conjugate, 1:6,000	Sigma-Aldrich	Cat# AP160P
Chemicals, peptides, and recombinant proteins
DMEM, no glucose	Gibco	Cat# 11966025
BenchMark fetal bovine serum	GeminiBio	Cat# 100-106
Opti-MEM	Gibco	Cat# 31985070
0.05% Trypsin-EDTA	Gibco	Cat# 25300054
Tris base	Sigma-Aldrich	Cat# 252859
Acrylamide	Sigma-Aldrich	Cat# A8887
Bis-acrylamide (N,N′-methylenebisacrylamide)	Sigma-Aldrich	Cat# M7279
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	Cat# L3771
Ammonium persulfate (APS)	Sigma-Aldrich	Cat# 248614
N,N,N′,N′-tetramethylethylenediamine (TEMED)	Sigma-Aldrich	Cat# T9281
HEPES	Sigma-Aldrich	Cat# H3375
Potassium chloride	Sigma-Aldrich	Cat# 529552
Magnesium chloride	Sigma-Aldrich	Cat# 208337
Sodium chloride	Sigma-Aldrich	Cat# S9888
Dithiothreitol	Thermo Scientific	Cat# R0861
Sucrose	Sigma-Aldrich	Cat# S8501
Bovine serum albumin	Sigma-Aldrich	Cat# 10735078001
ATP	Sigma-Aldrich	Cat# A1852
GTP	Sigma-Aldrich	Cat# G8877
Digitonin	Sigma-Aldrich	Cat# D141
Glycerol	Thermo Scientific Chemicals	Cat# A16205-AP
Bromophenol blue dye	Bio-Rad	Cat# 1610404
Glycine	Sigma-Aldrich	Cat# G8898
Methanol	Thermo Scientific Chemicals	Cat# 423955000
Isopropanol	Thermo Scientific Chemicals	Cat# 383910025
Triton X-100	Sigma-Aldrich	Cat# T8787
Tween 20	Sigma-Aldrich	Cat# P9416
Non-fat dry milk	LabScientific	Cat# M0841
Lipofectamine 2000	Invitrogen	Cat# 18324012
2′3′ cGAMP	InvivoGen	Cat# tlrl-nacga23-02
Coomassie brilliant blue G250	Sigma-Aldrich	Cat# 1.15444
Experimental models: Cell lines
HEK293T	ATCC	CRL-3216
V2 knockout HEK293T	Liu and Manley1	N/A
Recombinant DNA
pcDNA3-C-HA	Liu and Manley1	N/A
pcDNA3-STING-HA	Liu and Manley1	N/A
pCXS-3xFLAG-EV	Liu and Manley1	N/A
pCXS-3xFLAG-WDR33v2	Liu and Manley1	N/A
pCXS-3xFLAG-WDR33v3	Liu and Manley1	N/A

Materials and equipment

10% SDS separation gel solution

Reagent	Final concentration	Amount
1.5 M Tris-Cl, pH 8.8	375 mM	1 mL
29% Acrylamide + 1% bis-acrylamide	10%	1.34 mL
10% SDS	0.1%	40 μL
10% APS	0.1%	40 μL
TEMED	0.025%	1 μL
Distilled Water	N/A	Up to 4 mL
Total	N/A	4 mL

Make fresh just before use. Store distilled water and Tris-Cl stock solution at 20°C–25°C. Store all other stock solutions at 4°C. 10% APS is stable for 6 months at 4°C.

SDS stacking gel solution

Reagent	Final concentration	Amount
0.5 M Tris-Cl, pH 6.8	125 mM	500 μL
29% Acrylamide + 1% bis-acrylamide	4.5%	300 μL
10% SDS	0.1%	20 μL
10% APS	0.1%	20 μL
TEMED	0.05%	1 μL
Distilled Water	N/A	Up to 2 mL
Total	N/A	2 mL

Make fresh just before use. Store all stock solutions as indicated above.

10% native separation gel solution

Reagent	Final concentration	Amount
1.5 M Tris-Cl, pH 8.8	375 mM	1 mL
29% Acrylamide + 1% bis-acrylamide	10%	1.34 mL
10% APS	0.1%	40 μL
TEMED	0.025%	1 μL
Distilled Water	N/A	Up to 4 mL
Total	N/A	4 mL

Make fresh just before use. Store all stock solutions as indicated above.

CRITICAL: Do not add SDS in native separation gel solution.

Native stacking gel solution

Reagent	Final concentration	Amount
0.5 M Tris-Cl, pH 6.8	125 mM	500 μL
29% Acrylamide + 1% bis-acrylamide	4.5%	300 μL
10% APS	0.1%	20 μL
TEMED	0.05%	1 μL
Distilled Water	N/A	Up to 2 mL
Total	N/A	2 mL

Make fresh just before use. Store all stock solutions as indicated above.

CRITICAL: Do not add SDS in native stacking gel solution.

Digitonin permeabilization buffer

Reagent	Final concentration	Amount
500 mM HEPES, pH 7.2	50 mM	250 μL
1 M potassium chloride	100 mM	250 μL
30 mM magnesium chloride	3 mM	250 μL
1 mM Dithiothreitol (DTT)	0.1 mM	250 μL
850 mM sucrose	85 mM	250 μL
2% bovine serum albumin	0.2%	250 μL
10 mM ATP	1 mM	250 μL
1 mM GTP	0.1 mM	250 μL
Digitonin (20 mg/mL)	10 μg/mL	1.25 μL
Distilled water	N/A	498.75 μL
Total	N/A	2.5 mL

Make fresh ∼30 min before use. Store small aliquots of DTT, ATP, GTP, and digitonin at −20°C. Avoid freeze-thaw cycles of ATP and GTP. Store all other stock solutions at 4°C.

Note: This volume is for two transfection conditions. Scale up proportionally if there are more experimental conditions.

2X non-reducing SDS sample buffer

Reagent	Final concentration	Amount
500 mM Tris-Cl, pH 6.8	138.9 mM	10 mL
10% SDS	4.4%	16 mL
Glycerol	11.1%	4 mL
Bromophenol blue dye	0.01%	3.6 mg
Distilled water	N/A	6 mL
Total	N/A	36 mL

Store at 20°C–25°C indefinitely.

Note: Glycerol is viscous. Always pipette glycerol slowly and carefully. Only submerge a small portion of the tip to the liquid. Remove the tip from the liquid only when no more liquid is entering the tip. Also dispense glycerol slowly until most liquid leaves the tip. Then pipette up and down multiple times to wash out any residue glycerol in the tip.

10X SDS running buffer

Reagent	Final concentration	Amount
Tris base	250 mM	30 g
Glycine	1.92 M	144 g
SDS	1%	10 g
Distilled Water	N/A	Up to 1 L
Total	N/A	1 L

Store at 20°C–25°C indefinitely.

1X transfer buffer

Reagent	Final concentration	Amount
Tris base	25 mM	6.04 g
Glycine	192 mM	28.8 g
Methanol	10%	200 mL
Distilled Water	N/A	Up to 2 L
Total	N/A	2 L

Store at 4°C for several months.

1X native lysis buffer

Reagent	Final concentration	Amount
200 mM HEPES, pH 7.0	20 mM	150 μL
250 mM sodium chloride	25 mM	150 μL
Glycerol	10%	150 μL
Triton X-100	1%	15 μL
Distilled water	N/A	1035 μL
Total	N/A	1.5 mL

Make fresh just before use.

Note: Glycerol must be pipetted carefully and slowly. See the note from “2X non-reducing SDS sample buffer” above. Triton X-100 is also viscous. Pipette Triton X-100 in the same way as glycerol.

1X native running buffer

Reagent	Final concentration	Amount
Tris base	25 mM	3.02 g
Glycine	192 mM	14.4 g
Distilled Water	N/A	Up to 1 L
Total	N/A	1 L

Store at 4°C indefinitely.

1X G250 running buffer

Reagent	Final concentration	Amount
Tris base	25 mM	3.02 g
Glycine	192 mM	14.4 g
Coomassie brilliant blue G250	0.02%	0.2 g
Distilled Water	N/A	Up to 1 L
Total	N/A	1 L

Store at 4°C indefinitely.

Wash buffer

Reagent	Concentration	Amount
1X PBS	1X	1 L
Tween 20	0.1%	1 μL
Total	N/A	1 L

Store at 20°C–25°C indefinitely.

Note: Tween 20 is viscous. Pipette Tween 20 in the same way as glycerol and Triton X-100 as described above.

Blocking buffer

Reagents	Concentration	Amount
Non-fat dry milk	4%	2 g
Wash buffer	N/A	Up to 50 mL
Total	N/A	50 mL

Store at 4°C for one week.

Step-by-step method details

Express STING in HEK293T cells

Timing: 3 days

In this step, we express STING in HEK293T cells by plasmid transfection, because they do not express endogenous STING.¹² This feature of HEK293T cells makes them an excellent model for functional studies of different STING mutants.

• 1.Passage a confluent 10-cm dish of HEK293T cells as described in Steps 11-16 in the “preparation of HEK293T cells” section above.
• 2.Measure cell concentration.

Note: Use a hemocytometer or an automatic cell counter. We use LUNA-II automated cell counter (Logosbio) and follow the manufacturer's instructions. We typically obtain 1.5–2 million cells per mL.

• 3.
  Seed cells into a 12-well plate.

  • a.
    Seed 0.3 million cells per well.
  • b.
    Add additional DMEM to the seeded wells to make 2 mL.
  • c.
    Pipette up and down on the well to mix (Figure 2A).

Note: One well is for empty vector (EV) and the other is for STING transfection. At a later time after transfection, you will split one well of a 12-well plate into two wells of a 24-well plate, one for cGAMP treatment and one as a mock control. Increase the number of seeding wells if there are more experimental conditions (e.g. with or without co-expression of a putative STING regulator). You may optimize the number of cells for seeding such that the cells reach 50–70% confluency on the date of transfection.

• 4.Incubate the seeded plates in a 37°C humidified CO₂ incubator.
• 5.
  24 h after cell seeding, prepare DNA-Lipofectamine mixes.

  • a.
    Dilute 50 ng pcDNA3-STING-HA (STING plasmid) and pcDNA3-C-HA (EV) separately in 100 μL Opti-MEM in two 1.5 mL tubes.

    Note: Perform transfection in tissue culture hood.

    CRITICAL: We found that STING-HA is expressed strongly in HEK293T cells. 50 ng plasmid DNA produces physiological expression levels. Using more plasmid might result in unwanted transfection artefacts.

  • b.
    Mix by tapping the tubes 5-10 times. Spin at maximum speed for ∼10 s.
  • c.
    Prepare two tubes each containing 100 μL diluted Lipofectamine 2000 in Opti-MEM (1 μL Lipofectamine in 99 μL Opti-MEM).

    CRITICAL: First add 99 μL Opti-MEM to empty tubes, then add 1 μL Lipofectamine directly to Opti-MEM. Never allow Lipofectamine to contact the tube wall directly.

  • d.
    Mix by tapping the tubes 5-10 times. Spin at maximum speed for ∼10 s.
  • e.
    Transfer all 100 μL diluted STING plasmid and EV separately to the two tubes containing diluted Lipofectamine.
  • f.
    Mix by tapping the tubes 30-40 times. Spin at maximum speed for ∼10 s.
  • g.
    Incubate the DNA-Lipofectamine mixtures at 20°C-25°C for 5–10 min.
• 6.Gently add the two 200 μL DNA-Lipofectamine mixtures dropwise to different areas of the two seeded wells. Rock the 12-well plate gently (Figure 2A).

CRITICAL: Be very careful when adding the DNA-Lipofectamine mixtures to HEK293T cells to avoid washing out the seeded cells.

• 7.Incubate the transfected cells in a 37°C humidified CO₂ incubator.

Note: No need to change medium after transfection for HEK293T cells.

• 8.
  24 h after transfection, the transfected cells should be confluent. Split the transfected cells. Troubleshooting 2.

  • a.
    Warm up DMEM and trypsin-EDTA.
  • b.
    Gently aspirate the media.
  • c.
    Add 200 μL trypsin-EDTA per well. Rock the plate to evenly distribute trypsin-EDTA.

    CRITICAL: Do not wash with PBS. Confluent HEK293T cells detach from the well very easily.

  • d.
    Incubate at 20°C–25°C for 1–3 min.
  • e.
    Add 800 μL DMEM to each well. Pipette up and down to dissociate the cells to single cells.
• 9.Measure the concentrations of the transfected cells.

Note: We typically obtain ∼1.2–1.5 million cells per well.

• 10.
  Seed cells into a 24-well plate (Figure 2A).

  • a.
    Seed 0.5 million STING-transfected cells into two wells.
  • b.
    Seed 0.5 million EV-transfected cells into two other wells on the same 24-well plate.

Note: There are a total of 4 wells on a 24-well plate. One well of STING-transfected cells will be the cGAMP-treated sample, while the other will be the mock control. This is also the case for EV-transfected cells.

• 11.Add sufficient DMEM to each well to make 500 μL. Pipette up and down to mix the cells in the wells.

CRITICAL: Make sure that cells are evenly distributed on the wells.

• 12.Incubate the cells in a 37°C humidified CO₂ incubator.

Figure 2.

Major steps for STING oligomerization detection in HEK293T cells

(A) HEK293T cells do not express endogenous STING, making them an excellent model to study STING functions. We first transfect HEK293T cells with STING plasmid. We then split the transfected cells for different treatment conditions.

(B) STING’s natural ligand, cGAMP, is not cell-permeable. The most effective way to introduce cGAMP into cells at a low concentration (4 μM) is by digitonin permeabilization. Alternatively, due to the presence of active cGAMP importers, cGAMP can also be added directly to the culture medium at a higher concentration (> 100 μM).

(C) Detect disulfide bond-linked STING oligomers by non-reducing SDS-PAGE with no reducing agents in the sample buffer. p-Monomer: phosphorylated STING monomer.

(D) Detect STING oligomers in native states, independent of disulfide bond linkage, by BN-PAGE. No SDS or reducing agents in the sample buffer.

Activate STING by cGAMP treatment

Timing: 4 h

In this step, we activate the exogenous STING in HEK293T cells with its native ligand, cGAMP,⁴ by digitonin permeabilization,¹⁵ because cGAMP is not cell-permeable. Directly adding cGAMP to the culture media can also activate STING, as active cGAMP importers have been identified,^16,17 but this requires a high cGAMP concentration, and STING activation levels tend to be low.

• 13.
  20 h after seeding the transfected cells, prepare the digitonin permeabilization buffers.

  • a.
    Make the digitonin permeabilization buffer as described in the “materials and equipment” section.
  • b.
    Warm up the buffer at 20°C–25°C for 30 min.
  • c.
    Split the 2.5 mL permeabilization buffer into two 1.25 mL aliquots.
  • d.
    Add 2.5 μL 2 mM cGAMP to one of the aliquots to make the cGAMP buffer.
  • e.
    Add 2.5 μL water to the other aliquot to make the mock buffer.

Note: The final cGAMP concentration is 4 μM.

• 14.Gently aspirate the media from the 24-well plate.
• 15.Carefully and slowly add 500 μL cGAMP buffer to one of the wells containing STING-transfected cells and one of the wells containing EV-transfected cells.

Note: These are the cGAMP-treated samples (Figure 2B).

CRITICAL: Make sure that no cells are washed out. If some cells are washed out, leaving large unfilled areas on the well, then drastic cell death will occur following permeabilization buffer treatment (independent of cGAMP), and the experiment will fail.

• 16.Repeat the above step with the mock buffer to make the control samples.
• 17.Incubate the treated cells in a 37°C humidified CO₂ incubator for 30 min. Troubleshooting 3.
• 18.Warm up DMEM during the 30-min incubation.
• 19.
  Change the media.

  • a.
    Carefully aspirate the permeabilization buffers.
  • b.
    Carefully and slowly add 500 μL DMEM to the wells (Figure 2B).

CRITICAL: Again, avoid washing cells out.

• 20.Incubate the cells in a 37°C humidified CO₂ incubator for 3 h.
• 21.Harvest the samples either for non-reducing SDS-PAGE or for BN-PAGE as described below.

Note: Non-reducing SDS-PAGE and BN-PAGE are two separate methods for detection of different aspects of STING oligomerization. These two methods require different sample preparations. Choose one of them to proceed. If both methods are of interest, perform one of the methods first, then repeat the above major steps for the other method. Alternatively, you may follow the two major steps above using two sets of samples.

Detect STING disulfide bond-mediated oligomerization by non-reducing SDS-PAGE

Timing: 2–3 days

Following activation by cGAMP, STING undergoes oligomerization that involves formation of intermolecular disulfide bonds.^1,11 It is thus possibly to detect STING disulfide bond-linked oligomers by non-reducing SDS-PAGE described in the steps below, in which we omit the reducing agents, such as DTT and beta mercaptoethanol, in the sample buffer.

Note: You may follow your lab’s western blot protocol, but you must omit reducing agents in the sample buffer. For demonstration purposes, we describe western blot procedures in the two major steps below for STING only. For proper comparison between experimental conditions, you must include a loading control, such as GAPDH or β-Actin.

• 22.Dilute 2X non-reducing SDS sample buffer in distilled water to make 1.5 mL 1X buffer.
• 23.
  Harvest the samples.

  • a.
    Place the 24-well plate on ice.
  • b.
    Aspirate media from the 24-well plate.
  • c.
    Add 300 μL 1X non-reducing SDS sample buffer directly to each well.
  • d.
    Mix and completely scrape cells off the wells using 1000 μL pipette tips.
  • e.
    Transfer the cell lysates to ice-cold 1.5 mL tubes.
  • f.
    Keep all samples on ice.

Note: You may harvest the samples on a lab bench. If you want to load a specific amount of protein, lyse the cells with an appropriate lysis buffer, then measure protein concentration after clearing the lysates by centrifugation. After normalizing the protein concentration across samples, add equal volume of 2X non-reducing sample buffer.

• 24.
  Process the samples.

  • a.
    While keeping the sample on ice, use a 1 mL syringe without a needle to draw up all 300 μL cell lysate.
  • b.
    Attach a 20G needle to the syringe.
  • c.
    Pass the lysate through the needle 12 times.
  • d.
    Repeat this for all samples.

CRITICAL: The cell lysates are viscous due to release of genomic DNA. Passing the samples through a needle can shear genomic DNA to reduce viscosity, ensuring the correct volume of samples can be loaded onto a gel. Avoid making bubbles. Do not boil the samples.

Note: You may take aliquots from the processed samples, and add enough 1 M DTT such that the final DTT concentration is 100 mM to make reduced versions of the same samples. Skip Step 24 if you lyse the cells with a lysis buffer.

Pause Point: The processed samples can be stored at −80°C for several months.

• 25.Warm up the samples at 20°C–25°C for 15–20 min.

Note: Boil the reducing samples for 5 min.

• 26.
  Perform SDS gel electrophoresis (Figure 2C).

  • a.
    Assemble the gel electrophoresis apparatus with the SDS-PAGE gel.

    Note: Different electrophoresis apparatuses have different designs. We use Mini-PROTEAN Tetra Cell (Bio-Rad) and follow manufacturer’s instructions.

  • b.
    Fill the apparatus with 1X SDS running buffer diluted in distilled water.
  • c.
    Wash the gel wells with SDS running buffer.
  • d.
    Load 12 μL sample per well onto the gel. Load pre-stained protein marker onto the two outermost lanes.

    Note: If you measure protein concentrations, load 10–50 μg.

  • e.
    Run the gel at 20°C–25°C at a constant 80 V for 20 min or until all samples enter the separation gel.

    Note: You may use precast gels, such as Novex Tris-Glycine gels (Invitrogen), follow the manufacturer's instructions.

  • f.
    Increase the voltage to 100 V and continue electrophoresis for 90 min or until the dye front reaches ∼1 cm from the bottom of the gel.
• 27.
  Perform wet transfer.

  • a.
    Open the two glass plates to extract the gel.
  • b.
    Assemble the transfer apparatus.

    Note: Different transfer apparatuses have different designs. We use Mini Trans-Blot Cell (Bio-Rad) and follow the manufacturer's instructions.

  • c.
    Use nitrocellulose membrane with a pore size of 0.45 μm.
  • d.
    Fill the apparatus with cold transfer buffer.
  • e.
    Transfer proteins at 4°C at 90 V for 90 min.
• 28.Remove the nitrocellulose membrane and incubate it in blocking buffer at 20°C–25°C for 30 min with gentle shaking.

Note: Store the used and unused blocking buffer at 4°C.

• 29.(Optional) Cut out the top, bottom, and the two sides of the membrane to remove regions that do not contain proteins or are outside of the protein marker range.

Note: These regions of the membrane do not provide useful or reliable information. Cutting these regions out reduces the size of the membrane, conserving downstream reagents such as antibodies.

• 30.Incubate the membrane with mouse anti-HA primary antibody diluted in blocking buffer at 1:10000 at 4°C for 16–20 h with gentle shaking.

Note: Optimize the dilution factor for your antibody. You can dilute the antibody in the blocking buffer used in Step 28. Use the remaining blocking buffer for secondary antibody dilution the next day. Store the used diluted primary antibody at 4°C for no more than a week. You can reuse diluted primary antibody multiple times.

• 31.Wash the membrane with wash buffer for 5 min with gentle shaking.
• 32.Repeat Step 31 two more times.
• 33.Incubate the membrane with rabbit anti-mouse HRP-conjugated secondary antibody diluted in blocking buffer at 1:6000 at 20°C–25°C for 1 h with gentle shaking.

Note: Optimize the dilution factor for your antibody. Do not reuse diluted secondary antibody.

• 34.Repeat Step 31 three times.
• 35.Incubate the membrane with ECL and detect chemiluminescence signals. Troubleshooting 4.

Note: You may detect chemiluminescence signals by X-ray films or digital imagers. We use the ChemiDoc MP Imaging System (Bio-Rad) and follow the manufacturer's instructions.

Detect overall STING oligomerization by BN-PAGE

Timing: 2–3 days

Our previous study suggests that not all STING oligomers are formed by intermolecular disulfide bonds.¹ Since STING is a transmembrane protein, BN-PAGE is the most appropriate method to detect STING oligomers without disulfide bond linkage.¹³

CRITICAL: Handle samples on ice or at 4°C for all steps described in this section.

• 36.
  Harvest the samples.

  • a.
    Aspirate media from the 24-well plate.
  • b.
    Add 300 μL native lysis buffer directly to each well.
  • c.
    Mix and completely scrape cells off the wells using 1000 μL pipette tips.
  • d.
    Transfer the cell lysates to ice-cold 1.5 mL tubes.
  • e.
    Rotate the samples for 15 min. Centrifuge the lysates at maximum speed for 5 min.
  • f.
    Keep the supernatants.
  • g.
    Add 7.5 μL 5% Coomassie brilliant blue G250 to each sample.

Note: You may measure and normalize protein concentrations before adding G250. The detergent : G250 dye ratio should be 8:1¹³ (the native lysis buffer contains 1% Triton X-100).

CRITICAL: Do not boil samples.

Pause Point: You may store the samples at −80° for one day, but we recommend using them immediately.

• 37.
  Perform BN gel electrophoresis at 4°C (Figure 2D).

  • a.
    Assemble the gel electrophoresis apparatus with the BN-PAGE gel.

    Note: Different electrophoresis apparatuses have different designs. We use Mini-PROTEAN Tetra Cell (Bio-Rad) and follow manufacturer’s instructions.

  • b.
    Fill the inner chamber with G250 running buffer.
  • c.
    Fill the outer chamber with native running buffer.
  • d.
    Load 12 μL sample per well onto the gel.

    Note: If you measure protein concentrations, load 10–50 μg. Do not use protein ladders containing SDS. You may use bovine serum albumin, which naturally form different oligomers, as molecular weight standards.¹⁸

  • e.
    Run the gel at 80 V for 20 min.
  • f.
    Stop electrophoresis.
  • g.
    Remove all G250 running buffer from the inner chamber.
  • h.
    Refill the inner chamber with native running buffer.
  • i.
    Resume electrophoresis at 100 V until the dye front reaches ∼1 cm from the bottom of the gel.

    Note: Protein migration could be slow. You may increase the voltage.

• 38.Perform wet transfer, blocking, primary/secondary antibody incubation, and ECL detection as described in Steps 27-35 above. Troubleshooting 5.

Note: The gel/membrane will look blue after electrophoresis/transfer. The excessive G250 dye does not appear to interfere with downstream procedures, and the membrane will mostly be distained as it undergoes buffer incubation and washing.

Expected outcomes

For non-reducing SDS-PAGE, we detect both monomeric (∼37 kDa) and dimeric (∼75 kDa) STING, in both mock and cGAMP-treated samples¹ (Figure 3A). Following cGAMP treatment, STING undergoes phosphorylation at Ser366.⁵ Phosphorylated STING monomers appear as bands immediately above the monomer bands (Figure 3A). Higher-order STING oligomers, such as hexamers, should be more prominent in the cGAMP-treated samples¹ (Figure 3A). We also include reduced samples in this experiment, in which only STING monomers and phosphorylated STING monomers are detectable, indicating that STING oligomers indeed involve disulfide bond formation (Figure 3A). Our protocol also permits co-expression of STING regulators. In our previous study, we report that the ∼38 kDa isoform of WDR33 (V2) inhibits STING oligomerization, while the ∼30 kDa isoform (V3) increases all STING levels.¹ When we co-express these two proteins with STING, changes in STING disulfide bond-mediated oligomerization are rapidly discernible (Figure 3B).

Figure 3.

Expected outcomes of STING oligomerization by non-reducing SDS-PAGE and BN-PAGE

(A) By non-reducing SDS-PAGE, STING monomers and dimers are always detectable. STING hexamers and phosphorylated STING monomers appear only in cGAMP-treated cells. STING dimers and hexamers are not detectable after DTT addition, confirming that STING oligomerization can involve disulfide bond formation. EV: empty vector; ST: STING; p-Monomer: phosphorylated STING monomer.

(B) This protocol also allows co-expression of STING regulators. In our previous study,¹ we found that two non-canonical isoforms of human WDR33, termed V2 and V3, regulates STING oligomerization. Co-expression of V2 with STING inhibits STING oligomerization, while co-expression of V3 with STING increases overall STING protein levels, independent of cGAMP stimulation.

(C) By BN-PAGE, STING oligomers are more prominent following cGAMP treatment than by non-reducing SDS-PAGE, suggesting that not all STING oligomers are linked by disulfide bonds. There are significantly lower levels of STING oligomers in V2 knockout cells than in wild-type cells, due to formation of insoluble STING aggregates in the absence of V2¹. WT: wild type; KO: V2 knockout. Figure 3C reprinted with permission from Liu and Manley, 2024.¹

For BN-PAGE, we detect STING mostly as dimers and higher-order oligomers that may appear as smears^1,11 (Figure 3C). We also detect low levels of STING oligomers in mock samples, but we observe most productive STING oligomerization in cGAMP-treated cells (Figure 3C). We also perform BN-PAGE to study STING oligomerization in V2 knockout cells. We observe significantly lower levels of soluble STING oligomers in V2 knockout cells, reflecting strong formation of insoluble STING aggregates¹ (Figure 3C).

Limitations

This protocol describes experiments using exogenous STING in HEK293T cells. While HEK293T cells are a great model to study STING functions and regulation,^1,5,7,11,12 strong expression levels of STING in these cells make this model susceptible to transfection artefacts. We suggest 50 ng of plasmid based on our experience to limit accumulation to near physiological levels, but readers should test different plasmid amounts. To maximize cell viability following digitonin permeabilization, we perform experiments on 100% confluent HEK293T cells. It is possible that cell physiology changes under this confluency. Alternatively, you may treat transfected HEK293T cells at low confluency directly with a high concentration of cGAMP, but STING activation tend to be lower, making STING oligomer detection more difficult. We suggest lysing cells directly in sample buffers to minimize sample manipulation and to perform electrophoresis immediately after harvest, but we cannot rule out potential STING oligomer dissociation and/or formation in the sample buffers.

Troubleshooting

Problem 1

SDS/native separation gel is leaking or not solidifying.

Potential solution

• •If leakage occurs, make sure that the spacer and top plates are properly clamped together. You may use parafilm or tape to seal the two sides and the bottom of the plates.
• •10% APS and/or TEMED are not added to the solutions. Make sure they are added, as they are required for PAGE gel solidification.
• •10% APS stock is degraded. Make fresh 10% APS.
• •Temperature is too low. Cast the gels in a warmer room. Different from agarose gels, PAGE gels solidify more efficiently at higher temperatures.

Problem 2

Cells are not confluent 24 h post-transfection.

Potential solution

• •Cells have been in culture for too long. Start a new HEK293T culture.
• •Cells are growing too slowly. Increase the number of cells for seeding.
• •Cell density is too low at the time of transfection. Increase the number of cells.
• •Too much DNA/transfection reagent is used. Reduce the amount of DNA/transfection reagent.

Problem 3

Cells are dying after digitonin permeabilization.

Potential solution

• •There is cell loss during aspiration and/or addition of the buffer. Perform these steps very carefully.
• •Cell distribution is not even. Pipette the cell suspension up and down several times on the well and rock the plate to evenly distribute the cells.
• •Cells are growing too slowly. Increase the number of cells for seeding.
• •Cell density is too low before treatment. Increase the number of cells.
• •Alternatively, directly add cGAMP at a high concentration (>100 μM final concentration) to the culture media instead of using digitonin permeabilization.

Problem 4

For non-reducing SDS-PAGE, no bands are detectable or only monomers are detectable.

Potential solution

• •There is no STING expression or expression levels are too low. Start a new HEK293T culture or increase the amount of STING plasmid for transfection.
• •STING transfection is not successful. Follow Lipofectamine 2000 manufacturer’s protocol for plasmid transfection.
• •Proteins transfer is not successful. Make sure that the nitrocellulose membrane is firmly against the gel and the electrodes are connected properly during transfer.
• •Antibodies are incorrect. Make sure the primary antibody is against STING or the tag and the antigen of the secondary antibody is against the species in which the primary antibody is raised.
• •Antibody dilutions are suboptimal. Try different dilutions of both the primary and the secondary antibodies.
• •If only monomers are detectable, make sure that there are no reducing agents in the sample buffer. Do not boil the samples.

Problem 5

For BN-PAGE, no bands are detectable or only monomers are detectable.

Potential solution

• •Check the Potential solutions from Problem 4 above.
• •The amount of G250 is insufficient. G250 adds a net negative charge to proteins for electrophoresis. Make sure G250 concentration is 1/8 of that of detergent in the sample buffer.
• •The primary antibody may not detect STING in its native states. Use a STING antibody that detects native STING or use a tag antibody.
• •If only monomers are detectable, make sure that there are no reducing agents and/or SDS in the sample buffer. Always perform BN-PAGE at 4°C, and keep samples on ice or at 4°C.

Resource availability

Lead contact

• Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, James L. Manley (jlm2@columbia.edu).

Technical contact

• Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Lizhi Liu (ll3053@columbia.edu).

Materials availability

• This study did not generate new unique reagents.

Data and code availability

• •Original data for Figure 3 in the paper is available upon request.
• •This study did not generate/analyze dataset or code.

Acknowledgments

We thank Takahiro Seimiya for help with buffer recipes. L.L. was supported in part by the US NSF Graduate Research Fellowship (DGE-2036197). This work was supported by National Institutes of Health R35 GM118136 (to J.L.M.).

Author contributions

L.L. developed the protocol, performed the experiments, and wrote the manuscript. L.L. and J.L.M. edited the manuscript.

Declaration of interests

The authors declare no competing interests.