Np 40

Rabbit polyclonal antibodies against VP1, VP2, VP3, and VP4 were all generated from rabbit serum by immunization with corresponding purified protein, respectively. Horseradish peroxidase (HRP) labeled anti-rabbit (IgG) was purchased from KPL (Millford, MA, USA). Cell lysis buffer NP-40 (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40) used for western blotting was purchased from Beyotime (P0013F, Shanghai, China). Immunofluorescence secondary antibody fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit antibody (A0562) was purchased from Beyotime (Shanghai, China). The Exfect Transfection Reagent (T101-01/02) was purchased from Vazyme Biotechnology (Nanjing, China).

Cells were lysed in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP40 (Beyotime, Beijing, China), and protease inhibitors (TaKaRa, Dalian, China). Proteins in lysates were resolved by SDS–PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA for 1 h at room temperature and incubated with primary antibody recognizing β-catenin (Cell Signaling Technology, Danvers, MA, USA), Lamin B (Cell Signaling Technology, Danvers, MA, USA), or Gapdh (Boster, BM1623, Wuhan, China) at 4 °C overnight. Incubation with secondary horseradish peroxidase-labelled antibody was carried out for 1 h at room temperature. Protein bands were visualized by chemiluminescence using an Electro-Chemi-Luminescence (ECL) kit (Proteintech, Hubei, China) and exposed to X-ray film. Protein band intensities were quantified using Image-J software v. 1.45 (National Institutes of Health, NIH, Bethesda, MD, USA). The GAPDH was used as the internal control for total protein and cytoplasmic protein, and Lamin B1 was used as the internal control for nuclear protein.

Distal ileum tissues were homogenized for 20 min with an electrically powered instrument in a solution containing PMSF and NP-40 (Beyotime, China). Then, an enhanced BCA protein assay kit (Beyotime, China) was used for protein quantitation. The samples were mixed with loading buffer and boiled for 5 min to denature the proteins, which were then separated by SDS-PAGE and transferred onto PVDF membranes. After blocking nonspecific binding at room temperature in fast blocking buffer for 10 min, the membranes were incubated overnight at 4°C with specific antibodies against β-actin (1 : 10,000), Toll-like receptor 4 (TLR4, 1 : 1000), and nuclear factor-κB (NF-κB, 1 : 7500) (ZENBIO Biotechnology, China). After the membrane was washed thoroughly with TBST, it was incubated for 2 h with a horseradish peroxidase- (HRP-) conjugated secondary antibody. The membrane was thoroughly washed with TBST, and then, the bands were detected using enhanced hypersensitive electrochemiluminescence (ECL) western blotting detection reagents (ZENBIO Biotechnology, China) on a BIO-RAD ChemiDoc™ Touch Imaging System. Relative quantification of the levels of target proteins compared to those of β-actin was accomplished with ImageJ software.

Mouse monoclonal antibodies (mAbs) raised against GST (M0807-1) and β-actin (M1210-2) as well as rabbit polyclonal antibodies (pAbs) raised against Flag (0912-1), Myc (R1208-1), and GFP (SR48-02) were obtained from Huaan Biological Technology (Hangzhou, China). Mouse anti-Flag (F1804) and anti-Myc (05-419) mAbs were purchased from Sigma-Aldrich (St. Louis, MO, United States). Rabbit mAb raised against NPM1 (ab52644) was obtained from Abcam (Cambridge, MA, United States). Anti-Flag affinity resin (A2220) for immunoprecipitation was obtained from Sigma-Aldrich. NP-40 cell lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40] was obtained from Beyotime (P0013F; Shanghai, China). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG were obtained from KPL (Milford, MA, United States).

Cells were lysed with NP40 (Beyotime Biotechnology, China), and supplemented with proteasome inhibitor cocktail EDTA-free (Roche, Germany) and phosphatase inhibitor cocktail I/II (Topscience, Shanghai, China) 72 h–96 h post-transfection. WCE were collected after centrifugation at 14,000 × g. After protein concentration quantified, WCEs at equal concentration and volume were pre-cleaned by IgG of the same species as the indicated primary antibody and protein A/G agarose. The supernatant was immunoprecipitated over night at 4 °C with indicated primary antibodies and protein for following assays. The precipitated protein complexes were washed using NP40 lysis buffer at least five times before being separated on SDS-PAGE and immunoblotting by the indicated antibodies.
For ubiquitylation immunoprecipitation, cells were transfected by HA tagged ubiquitin (HA-ub) plasmids together with the other indicated plasmids. Before collecting the cell lysate, cells were treated with 20 μmol/L of MG-132 (Topscience, Shanghai, China) for at least 6 h. The following assays were performed as previous described. Antibodies applied in this study were listed in Supplementary Table 4.

The cells were lysed by NP-40 (Beyotime) containing 1/100 protease inhibitor Cocktail (MedChemExpress) for 15 min at 4 °C. After supersonic lysis and centrifugation, the supernatant was obtained and the protein concentration was measured using the Bicinchoninic Acid Protein Assay Kit (Thermo). Then the protein samples were mixed with SDS and denatured at 95 °C for 15 min. The samples were fractionated by 10% SDS-PAGE and transferred to Trans-blot membranes (Roche). The membranes were blocked by 5% skim milk for 1 h at room temperature and incubated with the primary antibodies as follows: WWP2 (1:1000; Abcam), KLF5 (1:1000; Proteintech), GAPDH (1:10,000; Proteintech), HIS (1:1000; Abcam), MYC (1:1000; Abcam), and PTEN (1:1000; Abclonal). After being incubated with secondary antibodies, the membranes were detected by ECL solution (Advansta).
Real-time PCR was carried out using fluorescent quantitation PCR mix from Vazyme in accordance with the manufacturer’s instructions. The primers were synthesized at Sangon. The primer sequences are listed in the Table S4.