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Method Article

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

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DOI:

10.3791/69321

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April 17th, 2026

 ,  ,  ,  ,  , 
1Department of Emergency Surgery, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Department of Medical Genetics, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology

In This Article

Summary

Ubiquitination is a critical regulator of intracellular protein functions. Here, we describe two cell lysis methods for detecting substrate protein ubiquitination levels in mammalian cells and compare their effectiveness in analyzing ubiquitination levels. Protein overexpression in HEK293T cells was induced by transient transfection, and ubiquitinated proteins were detected by western blot analysis.

Abstract

Ubiquitination, a post-translational modification, is a critical regulator of intracellular protein function. Ubiquitination modulates protein functions by promoting proteasomal degradation or altering subcellular localization through ubiquitin chain-dependent signaling. Here, we describe two cell lysis methods for detecting SMAD2 ubiquitination levels in HEK293T cells and compare their effectiveness in analyzing protein ubiquitination levels. Protein overexpression in cells was induced by transient transfection. The plasmids (HA-Ub, FLAG-SMAD2, and MYC-SMURF2) and transfection reagents were separately added to basal medium, mixed, and the mixture was added to the cells. Prior to harvest, MG132 was added to inhibit proteasomal degradation and enhance ubiquitinated protein accumulation. The primary divergence between the two experimental approaches is their cell lysis methods-the ice-bath method (performed at 4 °C) and the heat-treatment method (involving incubation at 95 °C), which substantially affects the efficiency of protein lysis. After cell lysis was completed, the cell lysate, agarose beads, and FLAG antibody were mixed and incubated at 4 °C overnight. Ubiquitinated proteins were then detected by western blot analysis. Before detecting ubiquitinated proteins, a light-chain antibody was used for secondary antibody incubation. Then, ubiquitination bands were detected. The results show that both the ice-bath method and the heat-treatment method can be used to detect ubiquitination levels, while the heat-treatment method may make it easier to detect ubiquitination of SMAD2. This study delineates and compares two cell lysis methods for measuring ubiquitination levels in mammalian cells, using SMURF2/SMAD2 as a model, to assist researchers in selecting more appropriate methods for detecting the ubiquitination levels of substrate proteins.

Introduction

Ubiquitination, an indispensable post-translational modification, is a critical regulator of intracellular protein functions1. The ubiquitin-proteasome system (UPS), which mediates the degradation of over 80% of proteins in eukaryotes, is composed of ubiquitin (Ub), E1 activating enzymes, E2 conjugating enzymes, E3 ligases, deubiquitylating enzymes (DUBs), and the 26S proteasome2. This system regulates protein turnover with high specificity, thereby controlling essential cellular processes such as cell cycle progression3, signal transduction4,5, and stress responses6,7. Ubiquitination is a multi-step enzymatic process. E1 hydrolyzes ATP to adenylate the C-terminus of Ub, forming a Ub-AMP intermediate. Then, the activated Ub is transferred from E1 to the active site cysteine of an E2 conjugating enzyme, forming a thioester bond. Finally, an E3 ligase recruits both the E2-Ub complex and a target protein, catalyzing the transfer of Ub from E2 to a lysine residue on the substrate8.

Ubiquitin can form diverse polyubiquitin chains through linkage at seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63) or Met1(M1)4. Each linkage type dictates distinct biological outcomes: K48-linked chains primarily target proteins for proteasomal degradation9, K63-linked chains regulate signaling and DNA repair10,11, and M1-linked chains are critical for innate immune responses12. Ubiquitination and deubiquitination are pivotal post-translational modification processes that govern diverse cellular events, ranging from protein degradation to signal transduction. For instance, in colorectal cancer, aberrant activation of MDM2-mediated p53 ubiquitination leads to excessive degradation of p53, impairing its ability to induce cell cycle arrest and apoptosis, thereby driving tumor progression and chemoresistance13,14. In hepatic ischemia-reperfusion injury, OTUD1 stabilizes NRF2 by removing its K48-linked polyubiquitination, thereby ameliorating intracellular oxidative stress levels15,16. A range of proteasome inhibitors, such as bortezomib, carfilzomib, and ixazomib, have progressively entered clinical practice for diseases such as multiple myeloma17,18,19.

Currently, there are numerous methods for detecting ubiquitinated proteins, including western blotting, radioactivity-based assays, and fluorescence assays20. However, in scientific research, the western blot remains the preferred method for the detection of protein ubiquitination due to its relatively rapid and straightforward protocol. Traditional methods for detecting ubiquitinated proteins are similar to co-immunoprecipitation (co-IP) technology: they use a lysis buffer to incubate and lyse cell pellets at low temperature to release proteins, then use antibodies to detect the ubiquitination of substrate proteins21,22. However, this experimental protocol often requires a large number of cells. Additionally, because the relatively mild lysis method at low temperature cannot completely disrupt protein interactions, this increases the likelihood of false positives in detected ubiquitination. Heating and the use of SDS can significantly disrupt protein structure, inducing their complete denaturation, thereby effectively reducing false positive results caused by protein-protein interactions23,24,25. Similarly, cells undergo sufficient lysis upon heating and SDS treatment, releasing more proteins and, correspondingly, reducing the amount of cells required for the experiment. In summary, both the ice-bath method and the heat-treatment method lyse cells and detect the ubiquitination levels of substrate proteins via antibody incubation. However, the heating method results in greater cell lysis and stronger protein denaturation.

TGF-β plays a dual role in the pathophysiological processes of various diseases26,27. SMAD2 is a critical downstream transcription factor of TGF-β. SMURF2-mediated ubiquitination of SMAD2 targets it for proteasomal degradation, thereby fine-tuning TGF-β signaling intensity28. This study delineates and compares two cell lysis methods for measuring ubiquitination levels in mammalian cells, using SMURF2/SMAD2 as a model. Here, protein overexpression in HEK293T cells was induced by transient transfection with HA-Ub, FLAG-SMAD2, and MYC-SMURF2 plasmids. The cells were lysed using the ice-bath method and the heat-treatment method, respectively. Ubiquitinated proteins were detected by Western blot analysis. We found that both the ice-bath method and the heat-treatment method can be used to detect ubiquitination levels, and the heat-treatment method might make it easier to detect ubiquitination of SMAD2. This protocol provides a new alternative for detecting ubiquitinated proteins. Furthermore, for certain substrate proteins strongly associated with ubiquitination, the heat-treatment method achieves better protein lysis efficiency, thereby enabling easier detection of ubiquitination.

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Protocol

1. Preparation of lysis buffer and reagents

NOTE: Prepare all buffers at the indicated concentrations. Follow the manufacturer's instructions for reagent powders to avoid concentration discrepancies.

  1. Prepare lysis buffer containing 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM EDTA (pH 8.0), and 1% Triton X-100. Adjust the NaCl concentration between 50-150 mM if required. Prepare all buffers using double-distilled H₂O.
  2. Prepare working solution by supplementing lysis buffer with 1% protease inhibitor cocktail (without EDTA). Mix thoroughly and keep on ice until use.
  3. Prepare 10% SDS by dissolving 1 g of SDS powder in 10 mL of PBS. Mix until fully dissolved. Store at room temperature.
  4. Prepare 5× protein loading buffer containing 250 mM Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 0.5% bromophenol blue, and 5% β-mercaptoethanol. Dissolve components in double-distilled H2O. Mix thoroughly before use.
    NOTE: Handle SDS and β-mercaptoethanol in a fume hood while wearing appropriate protective equipment. Dispose of contaminated materials according to institutional chemical waste guidelines.

2. Preparation of cells

  1. Maintain HEK293T cells in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Incubate at 37 °C with 5% CO₂ under sterile conditions. Passage cells at approximately 90% confluency.
  2.  Seed cells in 6-well plates at 5 × 10⁵ cells per well. Pool cells from two wells to generate one sample.
  3. Prepare transfection mixtures to represent 10% of the total culture volume and use SMAD2 ubiquitination as the experimental model. Combine 1 µg of HA-Ub plasmid, 1 µg of FLAG-SMAD2 plasmid, and 0.5 µg of empty vector or MYC-SMURF2 plasmid in 100 µL of serum-free DMEM. In a separate tube, dilute 3.75 µL of transfection reagent in 100 µL of serum-free DMEM. Add the reagent mixture dropwise to the plasmid solution, incubate for 15 min at room temperature, and distribute evenly onto cells.
  4. Replace the transfection medium after 6-8 h with 2 mL of fresh complete DMEM per well. Incubate cells for 48 h.
  5. Treat cells with 5 µM MG132 for 12 h before harvesting.
    NOTE: Handle MG132 in a biosafety cabinet while wearing gloves. Dispose of contaminated materials according to institutional safety procedures.

3. Cell collection

  1. Harvest cells at 90-100% confluency (~3 × 10⁶ cells). Aspirate medium and wash each well with 1 mL of prechilled PBS. Remove PBS completely.
  2. Add 1 mL of PBS to each well and gently resuspend cells. Transfer the suspension to a 1.5 mL tube and place on ice.
  3. Centrifuge at 380 × g for 5 min at 4 °C. Remove the supernatant and retain the pellet.

4. Agarose bead preparation

  1. Transfer 50 µL of Protein A/G agarose beads per sample to a new tube. Wash beads 3x with 1 mL of lysis buffer. Centrifuge at 100 × g for 1 min at 4 °C between washes.
  2. Remove residual buffer without disturbing the pellet. Add 100 µL of working solution and 5 µL of anti-FLAG antibody. Incubate for 6 h at 4 °C with gentle rotation.

5. Cell lysis

  1. Ice-bath method
    1. Add 500 µL of prechilled working solution to the pellet. Incubate on ice for 30 min.
    2. Sonicate at 2 W using 4 s on/4 s off cycles for five repetitions. Keep samples on ice during sonication. Return samples to ice for the remainder of the incubation.
      ​NOTE: For weak protein interactions, reduce NaCl concentration to 50-75 mM and omit sonication.
  2. Heat-treatment method
    1. Add 45 µL of working solution to the pellet. Add 5 µL of 10% SDS while mixing continuously.
    2. Vortex briefly and heat at 95 °C until the suspension becomes transparent and fully liquefied. Invert intermittently to ensure uniform denaturation.
    3. Add 450 µL of working solution. Sonicate at 2 W using 4 s on/4 s off cycles for five repetitions while keeping samples on ice.
      NOTE: Ensure final SDS concentration remains <0.1%. Confirm complete lysis by verifying disappearance of visible pellet and uniform solution clarity.

6. Immunoprecipitation and ubiquitination detection

  1. Centrifuge lysates at 13,680 × g for 15 min at 4 °C. Transfer supernatant to a new tube.
  2. Reserve 60 µL of lysate and add 15 µL of 5× loading buffer. Heat at 95 °C for 15 min and label as input.
  3. Add 100 µL of antibody-coupled beads to the remaining lysate (final volume 540 µL). Incubate at 4 °C for 8-12 h with rotation.
  4. Centrifuge beads at 100 × g for 1 min at 4 °C and discard supernatant. Wash beads 3x with lysis buffer. Add 75 µL of 1× SDS loading buffer and heat at 95 °C for 15 min to elute proteins. Label as IP.
  5. Detect ubiquitinated proteins by western blot according to a standard JoVE protocol.
    NOTE: Refer to the western blot protocol29 for detailed transfer and detection procedures. Full-length uncropped blots with molecular weight markers are provided in Supplemental File 1.

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Results

As a "molecular messenger" in TGF-β signaling pathway, SMAD2, after being phosphorylated byTGF-β receptors, forms a complex with SMAD3 and SMAD4, translocates to the nucleus, and initiates the transcription of downstream target genes30. It is thus a key molecule linking extracellular signals to intracellular gene expression. Its degradation is mediated by SMURF2, an E3 ubiquitin ligase that catalyzes the polyubiquitination of the target protein, thereby targeting it for proteasomal degradation. Th...

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Discussion

This study presents and compares two detailed protein lysis protocols specifically optimized for detecting E3 ligase-mediated substrate ubiquitination. To ensure detectability, we used plasmid-based overexpression (e.g., tagged SMAD2, SMURF2, or Ub) in cells, which provided sufficient protein levels to visualize specific ubiquitination signals, critical for validating E3 ligase-substrate interactions in this study. Prior to cell harvesting, MG132, a potent proteasome inhibitor, was added to block the proteasome-mediated ...

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Disclosures

The authors have no conflicts of interest to declare.

Acknowledgements

This research study was supported by the National Natural Science Foundation of China, Nos. 82370647 (W.H.), Nos. 82170642 (J-X.Z), Nos. 81801923 (X-F.Z), Nos. 81670575 (J-X.Z), Nos.81570570 (W.H.) and Nos. 81070355 (J-X.Z), and the Program of HUST Academic Frontier Youth Team, Huazhong University of Science and Technology (2018QYTD02). The Open Foundation of Hubei Key Laboratory of Regenerative Medicine and Multi-disciplinary Translational Research (2022zsyx003). The National Key Research and Development Program of China (2024YFA1107601).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1.5 mL microtubeServicebioEP-150-M
100 mm TC-treated cultured dishServicebioCCD-100
6-well diskServicebioCCP-6H
Anti-FLAG antibodiesProteintech20543-1-APdilution used 1:200 for IP 1:10000 for WB
Anti-GAPDH antibodiesProteintechMA5-15738dilution used 1:10000
Anti-HA antibodiesProteintech51064-2-APdilution used 1:10000
Anti-IgG antibodiesCST#3900dilution used 1:200 for IP
Anti-MYC antibodiesProteintech60003-2-Igdilution used 1:10000
Anti-SMAD2 antibodiesCST#5339dilution used 1:200 for IP 1:2000 for WB
Bromophenol blueServicebioGC307006-5g
EDTA (pH=8.0)ServicebioG1207-1L
FBSABW
GlycerolSinopharm10010618
Heating/Cooling Dry BathGuocheng
HEK293T cellsATCCCRL-3216
High-glucose DMEMServicebioG4515-500ML
HRP-conjugated Goat anti-Mouse IgG Light Chain AbclonalAS062dilution used 1:5000
HRP-conjugated Mouse anti-Rabbit IgG Light Chain AbclonalAS061dilution used 1:10000
Lipo8000BeyotimeC0533
MG132AladdinM421264
MicrocentrifugeLabgicCF1524R
NaCl powderServicebioGC102004
PBS ServicebioG4202-500ML
pCMV-FLAG-SMAD2For mammalian expression
pCMV-HA-UbFor mammalian expression
pCMV-MYCFor mammalian expression
pCMV-MYC-SMURF2For mammalian expression
Penicillin-streptomycinServicebioG4003-100ML
Protease inhibitor cocktail without EDTATargetMolC0001
Protein A/G agarose beadsBeyotimeP2017
PVDF membraneVayzmeE801
SDS powderSolarbioS8010-500g
SDS-PAGE sysytemAervicebioBVE4-BVT4
SonicsUibra cell
Tris-HCl powderBiotoppedHST-0372
Triton X-100ServicebioGC204003-100ml
TrypsinServicebioG4021-100ML
Vortex mixerKindly donated by Vayzme
β-mercaptoethanolAladdinM301573

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Protein UbiquitinationSMAD2 UbiquitinationSMURF2 ModelCell Lysis MethodsHeat Treatment LysisIce Bath LysisWestern BlotHEK293T CellsProteasomal Degradation
Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model
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