Kc 5g5

WB was performed according to the previously reported methods, with some modifications [21 (link)]. Cells were harvested in RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) that was supplemented with complete protease inhibitors (Sigma, Darmstadt, Germany). Cell lysates were separated on 10% SDS-PAGE and blotted onto the NC membrane. The membrane was then blocked in 5% nonfat milk and incubated with the primary antibodies overnight at 4 °C in the following dilutions: anti-G3bp1 (1:3000, ab181150, Abcam, Cambridge, UK), anti-G3bp1 (1:3000, ab56574, Abcam, Cambridge, UK), anti-G3bp2 (1:3000, NBP1-82977, Novus biologicals, Abingdon, UK), anti-Ubiquitin (1:1000, sc-8017, Santa Cruz, Dallas, TX, USA), anti-HSP70 (1:10000, ab45133, Abcam, Cambridge, UK), anti-VCP (1:3000, ab109240, Abcam, Cambridge, UK), anti-LC3 (1:1000, A19665, ABClonal, Wuhan, China), and anti-GAPDH (1:10000, KC-5G5, Kangchen Biotech, Shanghai, China). After the membranes were washed with 0.1% PBST, HRP-conjugated anti-mouse (7076S, CST, Danvers, MA, USA) or anti-rabbit (7074S, CST, Danvers, MA, USA) antibody was used as the secondary antibody for 1 h at room temperature. The signals were detected by using the Western ECL Substrate (170-5060, Bio-Rad, Irvine, CA, USA). The images were visualized by using the Bio-Rad ChemiDoc XRS+ system.

The expression level of HNF4α protein was measured by western blotting as previously described [18 (link)]. The following antibodies were used: primary antibodies HNF4α (Cat. no. ab201460, 1 : 1000, Abcam) and GAPDH (Cat. no. KC-5G5, 1 : 8000, KangChen Bio-tech, Shanghai, China) and an HRP-conjugated secondary antibody (Cat. no. bs-0295G-HRP, 1 : 8000, Boster, Wuhan, China). Endogenous GAPDH was used as a loading control. The X-ray film of the internal reference control GAPDH and the target protein HNF4α band was photographed using a gel image scanning analysis system. The integrated optical density value of each band was measured by using the gray-scale analysis software Image-Pro Plus 6.0. The optical density ratio of the target protein/internal reference protein is the relative expression of the target protein.