Anti hnf4α

Tissues or cultured cells were lysed with RIPA lysis buffer with protease inhibitors and phosphatase inhibitors (Beyotime, Shanghai, China) using standard methods. After centrifugation and quantification, 25 µg of protein was loaded for blotting with the indicated specific antibodies. A NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific, Waltham, USA) was used to isolate nuclear and cytoplasmic fractions according to the manufacturer’s protocol. The following antibodies were used: anti-Sox9 (1:1000, Abcam, Cambridge, UK), anti-HNF4α (1:2000, Abcam, Cambridge, UK), anti-TERT (1:1000, Novus, Colorado, USA), anti-E-Cadherin (1:1000, Abcam, Cambridge, UK), anti-Vimentin (1:1000, CST, Danvers, USA), anti-CK19 (1:1000, Abcam, Cambridge, UK), anti-LGR5 (1:1000, Abcam, Cambridge, UK), anti-Epcam (1:1000, Abcam, Cambridge, UK), anti-P65 (1:1000, CST, Danvers, USA), anti-p-P65 (1:1000, CST, Danvers, USA), anti-Bcl3 (1:100, Abcam, Cambridge, UK), anti-Histone H3 (1:1000, Proteintech, Rosemont, USA), anti-YAP1 (1:1000, CST, Danvers, USA), anti-CTGF (1:1000, Abcam, Cambridge, UK), anti-Cyr61 (1:1000, Abcam, Cambridge, UK), anti-His (1:5000, Proteintech, Rosemont, USA), anti-Flag (1:2000, Proteintech, Rosemont, USA), anti-Myc (1:1000, Proteintech, Rosemont, USA), anti-Ubiquitin (1:1000, CST, Danvers, USA), and anti-β-actin (1:4000, Bioworld, MN, USA).

Total protein was extracted by mixing cells with a radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors. Western blot analysis was carried out following standard procedures. The following antibodies were used for this experiment: anti-HDAC6 (1:1000, Cell Signaling Technology, #7558), anti-HNF4α (1:2000, Abcam, #92378), anti-MUC2 (1:4000, Abcam, #ab133555), anti-KLF4 (1:1000, Cell Signaling Technology, #4038), anti-CDX2 (1:1000, Cell Signaling Technology, #12306), and anti-β-actin (1:5000, Bioworld, #AP0060). The primary antibody dilution buffer, PREstain Protein Ladder and Western SuperSensitive Substrate were purchased from BioCytoSci (USA).

Total protein was extracted by mixing cells with radioimmunoprecipitation assay (RIPA) buffer (#P0013B, Beyotime, China), which was supplemented with protease and phosphatase inhibitors. Next, the proteins were separated by SDS-PAGE and transferred to NC membranes (#66485, Pall Inc., USA). The resulting membranes were incubated with 5% milk/TBST for 2 h at room temperature. Next, TBST was used to wash the membranes for 3 × 5 min, after which they were incubated with primary antibodies at 4 °C overnight. Finally, the membranes were incubated with HRP-conjugated secondary antibodies (#SAG10002, AntiProtech Inc., USA) after washing in TBST for 3 × 7 min. The following antibodies were used: anti-FXR (#187735, 1:200, Abcam, UK), anti-SNAI2 (#106077, 1:1000, Abcam, UK), anti-HDAC6 (#7558, 1:1000, Cell Signaling Technology, USA), anti-HNF4α (#92378, 1:2000, Abcam, UK), anti-CDX2 (#12306, 1:1000, Cell Signaling Technology, USA), and anti-β-actin (#AP0060, 1:5000, Bioworld, China). The primary antibody dilution buffer, PREstain Protein Ladder and Western SuperSensitive Substrate were purchased from BioCytoSci (#IC-8008, #IC-8001, China).