Phosphatase inhibitor cocktail 1 2

RIPA Lysis Buffer (Beyotime, Shanghai, China), Protease Inhibitor Cocktail (TOPSCIENCE), and Phosphatase Inhibitor Cocktail III (TOPSCIENCE) were used to extract proteins. SDS-PAGE was used to separate protein samples, which were then transferred to polyvinylidene fluoride membranes. HNF4G (1:3000, PP-N3224-00; R&D System, Minneapolis, MN, US), MAPK6 (1:3000, abs152150; Absin, Shanghai, China), p-MAPK6 (1:1000, AF7407; Affinity, Liyang, Jiangsu, China), Akt (1:3000, CY5561; Abways, Shanghai, China), p-Akt (1:1000, AP0637; Abclonal, Wuhan, China), GAPDH (1:3000, AG019; Beyotime) antibodies were used to detect the specific protein bands.

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.

Whole cell extract was isolated from 48 h post-transfection cells with RIPA lysis buffer (Beyotime Biotechnology, China) with proteasome inhibitor cocktail EDTA-free (Roche, Germany) and phosphatase inhibitor cocktail I/II (Topscience, Shanghai, China). After protein concentrations determinated by BCA protein assay Kit (Beyotime Biotechnology, China), the proteins were separated by SDS-PAGE, followed by western blotting with indicated antibodies. All the primary antibodies were listed in Supplementary Table 4. HRP conjugated Goat anti-Mouse/Rabbit antibody were used as secondary antibody (Boster, Wuhan, China) for chemiluminescence. Eventually, bands were visualized using SuperSignal™ West Atto Ultimate Sensitivity Substrate (ThermoFisher Scientific, USA) or Immobilob™ Western Chemiluminescent HRP Substrate (Millipore, USA). Antibodies applied in this study were listed in Supplementary Table 4. Uncropped western blot strips are provided in the supplementary material.