An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay

Summary

E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for preparing cells and transfecting plasmids. We detail the use of western blot to detect IGF2BP1 ubiquitination and a Cell Counting Kit-8 (CCK-8) assay to detect IGF2BP1 activation. This protocol is applicable to other proteins of interest.

For complete details on the use and execution of this protocol, please refer to Lin et al. (2021).¹

Graphical abstract

Highlights

• •Protocol to determine ubiquitination and activation of target proteins in vivo
• •Detection of ubiquitinated and activated proteins by western blot and CCK-8 assay
• •Adaptable to other interesting protein ubiquitination by E3 ubiquitin ligase
• •Easy and dependable way to assess protein ubiquitination levels in vivo

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

E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for preparing cells and transfecting plasmids. We detail the use of western blot to detect IGF2BP1 ubiquitination and a Cell Counting Kit-8 (CCK-8) assay to detect IGF2BP1 activation. This protocol is applicable to other proteins of interest.

Before you begin

Prepare high quality plasmid DNAs, cell lines and regents for ubiquitination assay. The protocol below describes the detailed procedure for ubiquitination analysis using a in vivo ubiquitination assay and protein activity evaluation using a CCK-8 assay. Here, we analyzed FBXO45-mediated IGF2BP1 ubiquitination by immunoprecipitation with Ni-NTA beads and IGF2BP1 activity after ubiquitination by CCK-8 assay. This protocol can also be applied to study the ubiquitination and activation of other proteins.

Ubiquitination is a post-translational modification that involves the covalent attachment of ubiquitin to one or more lysines of target protein to affect various cellular processes, including protein degradation, protein activity change, intracellular trafficking, and inflammatory response.^2,3,4 E3 ubiquitin ligases are key components of the ubiquitin proteasome system (UPS) that are critical in transferring ubiquitin to be conjugated to substrate lysines.^5,6,7 For this protocol, HEK293T, HepG2 and HCCLM3 cells were transiently transfected the plasmids of His-Ub and Flag-FBXO45 with HA-IGF2BP1, HA-IGF2BP1 (K190A), or HA-IGF2BP1 (K450A). For the in vivo ubiquitination assay, cells were harvested for immunoprecipitation with Ni-NTA beads and Western blotting was used to detect the ubiquitinated IGF2BP1 protein with the HA antibody. For substrate activation assay, to validate the activation of IGF2BP1 after polyubiquitination by FBXO45, IGF2BP1 wt- and its mutants-mediated cell proliferation was analyzed using a CCK-8 assay in HepG2 and HCCLM3 cells.

Preparation of endotoxin-free plasmids

Timing: 1 week (Construction), 2 days (Extraction)

• 1.
  Construction of recombinant expression plasmids.

  • a.
    Clone 3× Flag tagged FBXO45 and HA tagged IGF2BP1 into the pCDNA3.1 + vector for exogeneous expression, respectively.
  • b.
    Perform site-directed mutagenesis of IGF2BP1 plasmids using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, USA).
• 2.
  Transformation of recombinant expression plasmids.

  • a.
    Before starting, pre-warm a water bath/heat-block to 42°C. Pre-warm the ampicillin selective LB medium in a water bath at 37°C.
  • b.
    Dilute the plasmid with DEPC water or TE buffer to a concentration of 100 ng/μL.
  • c.
    Thaw DH5α competent cells fully on ice. Add 100 ng plasmids to the 50 μL thawed competent cells. Stir the solution gently to mix it well.
  • d.
    Incubate the mix solution on the ice for 30 min.
  • e.
    Give a heat shock to the cells by placing the reaction mix at 42°C for 90 s in the water bath/heat-block.
  • f.
    After heat shock, quickly transfer the cells onto the ice for 2 min.
  • g.
    Add 500 μL of pre-warmed LB broth medium, mix gently. Place the tubes in the shaker (200–250 rpm) at 37°C for 1–2 h.
  • h.
    Take 100–200 μL of the cell suspension using a pipette to an ampicillin selective LB dish. Distribute the solution evenly using a sterile spreader.
  • i.
    Incubate at 37°C in an incubator for 16–18 h.
• 3.
  Extraction of recombinant expression plasmids.

  • a.
    Pick a single colony with pipette tips and put it into 15 mL centrifuge tubes containing 100 μg/mL ampicillin LB medium (6 mL) and incubate in a shaker (200–250 rpm) at 37°C for 16 h.
  • b.
    Extract recombinant expression plasmids using a EndoFree Plasmid Midi Kit (Cwbiotech, China).
  • c.
    Measure the concentration of plasmids at A260 using a NanoPhotometer (IMPLEN, Germany). Plasmids can be stored at −20°C or −80°C.

Note: Pure DNA has an A₂₆₀/A₂₈₀ ratio of 1.8–2.0 in 10 mM Tris-HCl, pH 8.5.

Preparation of cell lines

Timing: 1 week

• 4.
  Thaw frozen cell lines.

  • a.
    Remove the cryovial containing the frozen cells from liquid nitrogen storage and place it into a 37°C water bath. Quickly thaw the cells (<1 min) by gently swirling the vial in the 37°C water bath.

    Note: “Quick thaw” in 37°C water bath by shaking vial rapidly in water.

  • b.
    Transfer the cells into the 6 mL pre-warmed DMEM complete growth medium (DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin) in a 15 mL centrifuge tube.
  • c.
    Centrifuge the cell suspension at approximately 200 × g for 5 min.
  • d.
    After centrifugation, carefully aspirate supernatant without disturbing the cell pellets.
  • e.
    Gently resuspend the cells in a total 8 mL fresh complete DMEM, and incubate cells at 37°C in 5% CO₂ incubator. Images are shown in Figure 1.

    CRITICAL: Get vial from Liquid Nitrogen-use gloves and face shield. ∗Make sure the cover is replaced properly!

• 5.
  Cells passage.

  • a.
    Passage HEK293T, HepG2 and HCCLM3 cells when 80%–90% confluent.
  • b.
    Remove culture medium and wash cells twice with 1X phosphate buffered solution (PBS) (approximately 5 mL).
  • c.
    Add 1 mL pre-warmed 0.25% Trypsin-EDTA to the dish. Gently rock the dish to completely cover the cells.
  • d.
    Incubate the dish in a 37°C incubator with a humidified atmosphere of 5% CO₂ for approximately 0.5–1.5 min.

    Note: The actual incubation time varies dependent on cell lines.

  • e.
    When the cells are detached and add 6 mL pre-warmed complete growth medium to inactivate trypsin.
  • f.
    Transfer the cells to a 15 mL conical tube and centrifuge at 200 × g for 5 min.
  • g.
    Remove culture medium and pass the cells to a 10 cm dish with a split ratio of 1: 3.

Figure 1.

Images of cells in culture

HEK293T, HepG2 and HCCLM3 cells were observerd under a microscope.

Preparation of reagent

Timing: 1 day

• 6.Prepare the required buffers (see materials and equipment below).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rat anti-HA (WB: 1/1000)	Roche	Cat. #:11867423001; RRID:AB_390918
Mouse anti-Flag (WB: 1/1000)	Sigma-Aldrich	Cat. #: F1804; RRID:AB_262044
Rabbit anti-GAPDH (WB: 1/5000)	Proteintech	Cat. #: 10494-1-AP; RRID:AB_2263076
Goat anti-mouse IgG HRP (WB: 1/5000)	Cell Signaling	Cat. #: 7076S; RRID:AB_330924
Goat anti-rabbit IgG HRP (WB: 1/5000)	Cell Signaling	Cat. #: 7074S; RRID:AB_2099233
Goat anti-rat IgG HRP (WB: 1/5000)	Cell Signaling	Cat. #: 7077S; RRID:AB_10694715
Chemicals, peptides, and recombinant proteins
MG-132, proteasome inhibitor	Selleck	Cat. #: S2619; RRID:N/A
Protease inhibitor cocktail	Bimake	Cat. #: B14012; RRID:N/A
Triton X-100	Sigma-Aldrich	Cat. #: T8787; RRID:N/A
Lipofectamine® 2000 Reagent	Invitrogen	Cat. #: 11668019; RRID:N/A
Ni-NTA Agarose	Qiagen	Cat. #: 30210; RRID:N/A
Critical commercial assays
QuikChange Lightning Site-Directed Mutagenesis Kit	Agilent	Cat. #: HB-8065; RRID:N/A
EndoFree Plasmid Midi Kit	Cwbiotech	Cat. #: cw2105s; RRID:N/A
Experimental models: Cell lines
HEK293T cells	ATCC	Cat. #: CRL-11268; RRID:CVCL_0063
HepG2 cells	ATCC	Cat. #: HB-8065; RRID:CVCL_0027
HCCLM3 cells	BeNa Culture Collection	Cat. #: 342335; RRID:CVCL_6832
Recombinant DNA
pcDNA3.1-FBXO45-3×FLAG	Lin et al. (2021)	RRID:N/A
pcDNA3.1-IGF2BP1-HA	Lin et al. (2021)	RRID:N/A
pcDNA3.1-IGF2BP1 (K190A)-HA	Lin et al. (2021)	RRID:N/A
pcDNA3.1-IGF2BP1 (K450A)-HA	Lin et al. (2021)	RRID:N/A
pcDNA3.1-Ub-6×His	Lin et al. (2021)	RRID:N/A
Software and algorithms
Image Lab	Bio-Rad Laboratories	https://www.bio-rad.com/
Other
Ultrasonic homogenizer	Scientz	Cat. #: Scientz-IID
NanoPhotometer	Implen	Cat. #: N60 Touch
Microplate reader	Thermo Scientific	Cat. #: VARIOSKAN FL

Materials and equipment

Reagents for ubiquitination

Buffer A (pH = 8.0)

Reagent	Final concentration	Amount
Guanidinium-HCl (8 M stock)	6 M	100 mL
pH8.0 Tris-HCl (1 M stock)	10 mM	1.33 mL
Na2HPO4/NaH2PO4 (1 M stock)	100 mM	13.3 mL
NaOH (1 M stock)	N/A	Require
HCl (2 M stock)	N/A	Require
ddH2O	N/A	Up to 133 mL
Total	N/A	133 mL

Note: Store at 20–25°C for up to 1 year.

CRITICAL: Concentrated hydrochloric acid is extremely volatile and corrosive. Sodium hydroxide is a highly corrosive alkali. Always wear personal protective equipment, including face mask and gloves. Please operate in a fume hood.

Buffer A1-lysis buffer (PH = 8.0)

Reagent	Final concentration	Amount
Buffer A	N/A	9.95 mL
Imidazole (1 M stock)	50 mM	0.05 mL
Total	N/A	10 mL

Note: The lysis buffer A1 can be stored at 4°C and should be freshly prepared to avoid loss of efficacy.

Buffer A2-wash buffer (PH = 8.0)

Reagent	Final concentration	Amount
Buffer A	N/A	9.9931 mL
β-mercaptoethanol (14.4 M stock)	10 mM	0.0069 mL
Total	N/A	10 mL

Note: The wash buffer A2 can be stored at 20–25°C and should be freshly prepared to avoid loss of efficacy.

CRITICAL: β-mercaptoethanol is a volatile toxic liquid and is absorbed through the skin. Please wear a face mask and gloves when using solutions in a fume hood.

Buffer B (pH = 8.0)

Reagent	Final concentration	Amount
Urea (10 M)	8 M	100 mL
pH8.0 Tris-HCl (1 M stock)	10 mM	1.25 mL
Na2HPO4/NaH2PO4 (1 M stock)	100 mM	12.5 mL
NaOH (1 M stock)	N/A	Require
HCl (3 M stock)	N/A	Require
ddH2O	N/A	Up to 125 mL
Total	N/A	125 mL

Note: Store at 20–25°C for up to 1 year.

Buffer B1-wash buffer (PH = 8.0)

Reagent	Final concentration	Amount
Buffer B	N/A	9.9931 mL
β-mercaptoethanol (14.4 M stock)	10 mM	0.0069 mL
Total	N/A	10 mL

Note: The wash buffer B1 can be stored at 20–25°C and should be freshly prepared to avoid loss of efficacy.

Buffer C (pH = 6.3)

Reagent	Final concentration	Amount
Urea (10 M)	8 M	100 mL
pH6.3 Tris-HCl (1 M stock)	10 mM	1.25 mL
Na2HPO4/NaH2PO4 (1 M stock)	100 mM	12.5 mL
NaOH (1 M stock)	N/A	Require
HCl (3 M stock)	N/A	Require
ddH2O	N/A	Up to 125 mL
Total	N/A	125 mL

Note: Store at 20–25°C for up to 1 year.

Buffer C1-wash buffer (PH = 6.3)

Reagent	Final concentration	Amount
Buffer C	N/A	9.9731 mL
β-mercaptoethanol (14.4 M stock)	10 mM	0.0069 mL
Triton X-100	0.2%	0.02 mL
Total	N/A	10 mL

Note: The wash buffer C1 can be stored at 20–25°C and should be freshly prepared to avoid loss of efficacy.

Buffer C2-wash buffer (PH = 6.3)

Reagent	Final concentration	Amount
Buffer C	N/A	9.9831 mL
β-mercaptoethanol (14.4 M stock)	10 mM	0.0069 mL
Triton X-100	0.1%	0.01 mL
Total	N/A	10 mL

Note: The wash buffer C2 can be stored at 20–25°C and should be freshly prepared to avoid loss of efficacy.

Buffer elution (1×)

Reagent	Final concentration	Amount
pH6.8 Tris-HCl (0.2 M stock)	20 mM	1 mL
Glycerine	10%	1 mL
SDS	0.8% (W/V)	0.08 g
Bromophenol blue	0.1% (W/V)	0.01 g
β-mercaptoethanol (14.4 M stock)	720 mM	0.5 mL
Imidazole (1 M stock)	300 mM	3.03 mL
ddH2O	N/A	Up to 10 mL
Total	N/A	10 mL

Note: Aliquot and store at −20°C for up to 1 year.

CRITICAL: SDS may cause skin and respiratory irritation and should be handled in a fume hood. Please wear a face mask and gloves.

Reagents for western blot

SDS-PAGE separating gel (10%) 1.5 mm gel ×2

Reagent	Final concentration	Amount
30% Acr-Bis (29:1)	10%	5 mL
pH8.8 Tris-HCl (1 M stock)	0.38 mM	5.7 mL
10% SDS	0.1%	0.15 mL
10% APS	0.1%	0.15 mL
TDMED	0.04%	0.006 mL
ddH2O	N/A	Up to 15 mL
Total	N/A	15 mL

Note: Prepare the solution just before use.

SDS-PAGE stacking gel (5%) 1.5 mm gel ×2

Reagent	Final concentration	Amount
30% Acr-Bis (29:1)	5%	1 mL
pH6.8 Tris-HCl (1 M stock)	0.125 mM	0.75 mL
10% SDS	0.1%	0.06 mL
10% APS	0.1%	0.06 mL
TDMED	0.1%	0.006 mL
ddH2O	N/A	Up to 6 mL
Total	N/A	6 mL

Note: Prepare the solution just before use.

CRITICAL: Acrylamide monomer is a potent cumulative neurotoxin and is absorbed through the skin. Do not mouth pipette acrylamide solutions. Please wear a face mask and gloves when handling unpolymerized solutions in a fume hood. Moreover, TEMED is a volatile and a potent cumulative neurotoxin. Always wear personal protective equipment, including a face mask and gloves. Please operate in a fume hood.

Note: Volumes given are sufficient for small (8 cm × 10 cm × 1.5 mm) gel format (10 mL of monomer).

Alternatives: Pre-cast gels are also available, such as Bio-Rad Cat#4561036 (10%) or Beyotime Biotechnology Cat#P0012A.

2× loading buffer (PH = 6.8)

Reagent	Final concentration	Amount
Tris-base	15.2 g/L	1.52 g
SDS	4%	4 g
Glycerin	20%	20 mL
Bromophenol blue	0.1%	50 mg
β-mercaptoethanol (14.4 M stock)	2%	2 mL
NaOH (1 M stock)	N/A	Require
HCl (2 M stock)	N/A	Require
ddH2O	N/A	Up to 100 mL
Total	N/A	100 mL

Note: Aliquot and store at −20°C for up to 1 year.

Alternatives: 3.1 g Dithiothreitol (DTT) can be alternative to β-mercaptoethanol. Pay attention to operate DTT in a fume hood and wear a face mask and gloves. Pre-mix 5× loading buffer are also available, such as Beyotime Biotechnology Cat#P0286.

Blocking buffer

Reagent	Final concentration	Amount
Non-fat milk	5%	0.5 g
TBST buffer (1×)	N/A	10 mL
Total	N/A	10 mL

Note: Store at 4°C for up to 1week or −20°C for up to 1 month.

Step-by-step method details

Seeding and transfection cells

Timing: 1 h (Seeding), 8 h (transfection)

The following steps describe the detailed procedure to seed cells, and to efficiently transfect His-Ub and Flag-FBXO45 plasmids with HA-IGF2BP1, HA-IGF2BP1 (K190A), or HA-IGF2BP1 (K450A) plasmids by using Lipofectamine® 2000 Reagent.

• 1.
  Seed cells 16–18 h before transfection.

  • a.
    Follow the “before you begin” step 5 to seed cells.
  • b.
    Seed HEK293T cells into 10 cm dishes with 60% confluent for ubiquitination assay.
• 2.
  Transfect with plasmids in the next morning.

  • a.
    Cell morphology and density are observed under a microscope. Once the cells are 70% confluent, transfect them with the indicated plasmids.
  • b.
    Add pre-warmed 500 μL Opti-MEM in the tube A and tube B. Add indicated plasmids (Amount of plasmids refer to Table1 ) in tube A and mix well. Add 30 μL Lipofectamine® Reagent in tube B and mix gently.
  • c.
    Add diluted DNA of tube A to tube B and incubate DNA-lipid complex at 20°C–25°C for 5 min.
  • d.
    Add the mixture dropwise to cells. Change to complete culture medium after 6-8 h transfection.
  • e.
    After 72 h transfection, cells are harvested for ubiquitination in vivo assay (see troubleshooting 1, 2).

Table 1.

Dose of plasmid DNA and transfection reagent for 10 cm dish

	Plasmid DNA						Lipofectamine® 2000 reagent (μL)
	His-Ub (6 μg)	Flag-FBXO45 (8 μg)	HA-IGF2BP1 (6 μg)	HA-IGF2BP1 (K190A) (6 μg)	HA-IGF2BP1 (K450A) (6 μg)
Tube A1	+					Tube B1	30
Tube A2	+	+				Tube B2	30
Tube A3	+		+			Tube B3	30
Tube A4	+	+	+			Tube B4	30
Tube A5	+	+		+		Tube B5	30
Tube A6	+	+			+	Tube B6	30
	Opti-MEM®Medium 500 μL/tube

Assay for protein ubiquitination in vivo

Timing: 3 days

F-box/SPRY domain-containing protein 1 (FBXO45) is an atypical E3 ubiquitin ligase,⁸ and is critical in the UPS for specific proteins degradation, such as PAR4 (prostate apoptosis response protein 4),⁹ p73,¹⁰ and ZEB1.¹¹ Here, the following steps describe the detailed procedure to determine IGF2BP1 as a novel substrate of FBXO45. IGF2BP1 can be polyubiquitinated in an in vivo system containing FBXO45(E3), HA-IGF2BP1 (substrate), and His-Ub in HEK293T cells. Proteins were separated by SDS-PAGE, and ubiquitinated IGF2BP1 was detected by HA antibody.

• 3.
  MG132 treatment.

  • a.
    Add MG132 at final concentration of 10 μM, mix gently.
  • b.
    Incubate the dishes in a 37°C incubator for 3–4 h to block protein degradation before cell collection.

• 4.
  Cell collection, cell lysis and Ni-NTA beads pull down.

  • a.
    Before starting, pre-warm a water bath/heat block to 100°C.
  • b.
    Wash the cells twice with 5 mL of ice-cold 1XPBS.
  • c.
    Scrape cells off using a plastic scraper and transfer them into 1.5 mL EP tubes.

    Note: One portion of cells are used as Whole Cell Extract group. The other are used for ubiquitination group.

  • d.
    Centrifuge at 900 × g for 5 min at 4°C. Discard the supernatant and place the cell pellets on the ice.
  • e.
    For the Whole Cell Extract group, add 200 μL RIPA buffer on ice for 15 min and centrifuge at 14,000 × g for 10 min at 4°C.

    • i.
      Measure the concentration of proteins with a BCA protein assay kit (Beyotime, China).
    • ii.
      Mix 20–30 μg protein from each sample with 2× loading buffer and boil for 10–15 min at 100°C.
    • iii.
      Store whole extract proteins at −20°C until use.
  • f.
    For the ubiquitination group, add 1 mL ice-cold lysis buffer A1 to the pellet and pipette the mixture up and down to lyse the cells (see troubleshooting 3). Incubate lysis solution on the ice for 30 min.

    Note: This step is considerably critical. It is important to ensure cells completely lysis. If the cells are still viscous after blowing, Ultrasonic Homogenizer (SCIENTZ, China) can be used to disrupt the cells continually.

  • g.
    Add 50 μL Ni-NTA beads (Qiagen, Germany) to each sample. Incubate and shake the mixture gently at 2–8°C for 16 h.

    Note: Resuspend the bead slurry by vortexing or mixing before use.

• 5.
  Perform in vivo assay of protein ubiquitination.

  • a.
    Centrifuge the beads at 600 × g for 5 min at 4°C.
  • b.
    Discard the supernatant, the beads-protein complex is then washed as above (a) with buffer A1, B1, C1 and C2, respectively (see troubleshooting 4).

    Note: To reduce the loss of beads during the washing steps, we recommend to aspirate most of the supernatant with the pipette along the wall of the tube, and keep 50 μL of liquid at the bottom of the 1.5 mL tube.

  • c.
    After washing, the His∗6-tagged ubiquitinated proteins are eluted with 50 μL 1× elution buffer.
  • d.
    Boil for 5 min at 100°C, and centrifuge at 16,000 × g at 20–25°C for 1–2 min to collect solution, then store at −20°C freezer for ubiquitination analysis using Western blot.

    Pause point: After sample boiling, whole cell extract proteins (step 4e) and ubiquitination proteins (step 5d) can be stored at −20°C until SDS-PAGE.

• 6.
  Prepare SDS-PAGE gels.

  • a.
    Prepare the separation gel (10%). Mix in the following order as above. Pour gel, leaving ∼2 cm below the bottom of the comb for the stacking gel. Make sure to remove bubbles.
  • b.
    Layer the top of the gel with isopropanol to remove bubbles at the top of the gel.

    Note: In ∼30 min, the gel should be completely polymerized.

  • c.
    Remove the isopropanol and wash out the remaining traces of isopropanol with distilled water.
  • d.
    Prepare the stacking gel (5 %). Mix in the following order as above. Pour stacking gel on top of the separation gel.
  • e.
    Add combs to make wells. In ∼30 min, the stacking gel should become completely polymerized.
  • f.
    Clamp gel into apparatus, and fill buffer chambers with gel running buffer.
  • g.
    Load whole extract proteins (∼15–30 μg), ubiquitinated proteins (∼100–150 μg) and molecular mass protein markers into wells for separation by electrophoresis.

    Note: The Western blot loading amounts have to be sufficient for the detection of protein of interest.

• 7.
  Run gel and Protein transfer from gel to membrane.

  • a.
    Run the gels at 60–80 V constant voltage for stacking gel (30 min) and then 100–150 V for separating gel (1 h).
  • b.
    After electrophoresis, remove the gel from the electrophoresis apparatus and equilibrate it by soaking in 1× Transfer Buffer for 10 min.
  • c.
    When performing a wet transfer, the gel is first equilibrated in transfer buffer. The gel is then placed in the “transfer sandwich” (filter paper-gel-membrane-filter paper), cushioned by pads and pressed together by a support grid. The detailed procedure can follow Prisco’s protocol step 82.¹²
  • d.
    Run at 80 V constant voltage for 2 h or at 100 V for 90 min on the ice. Proteins are transferred from gel to PVDF membrane.

• 8.
  Detect proteins.

  • a.
    Prepare blocking buffer (refer to materials and equipment above) and block the membrane for 1 h at 20–25°C using blocking buffer.
  • b.
    Add a primary antibody diluted in 10 mL blocking buffer. Incubate for 16–18 h at 4°C (see troubleshooting 5).

    Alternatives: Membranes can also be incubated with shaking at 20–25°C for 1 h.

  • c.
    Wash the membranes three times with 1 X TBST buffer at 10 min for each time.
  • d.
    Add secondary antibody in 10 mL blocking buffer. Shake gently at 20–25°C for 1 h.
  • e.
    Wash the membranes three times with 1 X TBST buffer at 10 min for each time.
  • f.
    Add developing reagent on the membrane, monitoring development using ChemiDocTM XRS+. Image is analyzed using LabTM Software (Bio-Rad, USA). A typical IGF2BP1 ubiquitination blot is shown in Figure 2 (see troubleshooting 6).

Figure 2.

FBXO45 promotes IGF2BP1 polyubiquitination at the Lys190 and Lys450 sites

IGF2BP1 was polyubiquitylated by FBXO45, and K190 or K450 mutation partly blocked this effect. HEK293T cells were transfected with the indicated plasmids, followed by pull-down using Ni-NTA beads or direct Western blotting with the indicated antibodies. Data is reproduced from Lin et al.¹.

Assay for ubiquitinated protein activation analysis

Timing: 3 h

The following steps describe the procedure to validate whether FBXO45 increased IGF2BP1 activity via polyubiquitination at K190 and K450, the effect of FBXO45 and its mutants on cell proliferation was analyzed by CCK-8 assay.

• 9.
  Cell Proliferation Assay.

  • a.
    Seed HepG2 and HCCLM3 cells in 96-well plates at a density of 3,000 cells per well in 100 μL fresh DMEM medium for CCK-8 assay.
  • b.
    Transfect cells with the indicated plasmids (same step as shown in the step 2 section). Dose of plasmids and transfection reagents refer to Table 2 for CCK-8 assay.
  • c.
    After 48 h transfection, add 10 μL the CCK-8 solution to each well of 96-well pate.

    Note: Pay attention not to introduce bubbles in the well.

  • d.
    Incubate the plate in a 37°C incubator for 2–3 h.
  • e.
    Measure absorbance at 450 nm using a Microplate Reader (Thermo Scientific, USA). The results of cell proliferation are shown in Figure 3.

Table 2.

Dose of plasmid DNA and transfection reagent for 96-well

	Plasmid DNA					Lipofectamine® 2000 reagent (μL)
	Flag-FBXO45 (1 μg)	HA-IGF2BP1 (1 μg)	HA-IGF2BP1 (K190A) (1 μg)	HA-IGF2BP1 (K450A) (1 μg)
Tube A1					Tube B1	2
Tube A2	+				Tube B2	2
Tube A3		+			Tube B3	2
Tube A4	+	+			Tube B4	2
Tube A5	+		+		Tube B5	2
Tube A6	+			+	Tube B6	2
	Opti-MEM®Medium 35 μL/tube/row

Figure 3.

FBXO45 promotes IGF2BP1 cell proliferation at the Lys190 and Lys450 sites

(A and B) HepG2 and HCCLM3 cells were co-transfected with indicated plasmids for 48 h, followed by CCK-8 assay. Data are represented as the mean ± SEM, n = 6 (HCCLM3), n = 5 (HepG2), ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. Data is reproduced from Lin et al.¹.

Expected outcomes

For ubiquitination assay, we expect to detect the protein of IGF2BP1 ubiquitination increased by E3 ligase. As shown in Figure 2, FBXO45 enhanced IGF2BP1 polyubiquitination, while the K190A or K450A mutants significantly blocked this action.

For activation assay, we expect to detect IGF2BP1 activity increased by FBXO45 using CCK-8 assay. As shown in Figure 3, Wild-type IGF2BP1 dramatically promoted HCC proliferation, whereas K190A and K450A mutants lost its oncogenic activity.

Limitations

This protocol works well to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45 and can be applied to other proteins of interest. To better analyze the protein ubiquitination by E3 ubiquitin ligase using Ni-NTA pull-down protein ubiquitination in vivo assay. For IGF2BP1, we recommend using the same antibody and setup conditions used in this protocol. For other interest proteins, you will likely need to test several antibodies from different companies, to weak the conditions to get the best results.

But this protocol is based on transient overexpression of multiple plasmids in HEK293T cells. Overexpression of proteins using excess lipofectamine may increase cytotoxicity in some cell types, which might affect the results. Hence it is important to optimize the plasmid amounts used for transfection.

Troubleshooting

Problem 1

Cytotoxic effects after transfection observed (step 2e of step-by-step method details).

Potential solution

• •Too much Lipofectamine® 2000 Reagent may be used. Reduce or optimize amounts of transfection reagent for each cell line.
• •Cells are very sensitive to transfection. Always transfect cells around 70% confluency. Increase or optimize cell confluency for each cell line before transfection.

Problem 2

When to harvest cells after transfection (step 2e of step-by-step method details).

Potential solution

Transfection conditions for plasmids of different sizes are always different. Try different time gradients in pre-experiment and select the best transfection time periods to harvest cells.

Problem 3

HEK293T cells are lysed thoroughly in lysis buffer (step 4f of step-by-step method details).

Potential solution

Ultrasonic Homogenizer (SCIENTZ, China) can be used to disrupt the cells to reduce sample viscosity. Sonicate the mixture by using a lab probe sonicator. The detailed parameters are set in this study as follows: 100 W; 2 s ON, 5 s OFF, 6 cycles. Sonication conditions may vary depending on cell type and volume.

Problem 4

A lot of beads-protein complex is lost during washing (step 5b of step-by-step method details).

Potential solution

To reduce the loss of beads during the washing steps. We recommend aspirating the supernatant along the wall of the tube using a pipette and keeping 25–50 μL of liquid left at the bottom of tube.

Problem 5

High background of the Western blot membrane (step 8b of step-by-step method details).

Potential solution

• •Extend the blocking time by 1–2 h to reduce background.
• •The concentrations and incubation conditions (e.g., temperature and time) of the first and secondary antibodies should be tested in advance. High concentrations of antibodies may cause high background of membrane, optimize antibody concentrations followed by stringent washes.

Problem 6

No ubiquitination or weak ubiquitylation (step 8f of step-by-step method details).

Potential solution

• •Loading protein amounts should be sufficient. If possible, include a known substrate of E3 ligase as a positive control.
• •Prepare fresh assay buffer every time to avoid the loss of protein protection activity. Meanwhile, avoid multiple freeze thaw cycles which may reduce protein activity.
• •Low concentrations of primary antibody may cause weak bands, optimize antibody concentrations or change another primary antibody to improve the signal-to-noise ratio. Alternatively, increase the incubation time.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Chuan-Ming Xie (cmxie@tmmu.edu.cn or chuanming506@126.com ).

Materials availability

Plasmids used in this study are available by contacting the lead contact, Chuan-Ming Xie (cmxie@tmmu.edu.cn or chuanming506@126.com).

Data and code availability

This study did not generate new data and codes.