Bs 20596r

RIPA buffer (Beyotime) was utilized for extracting total protein from serum and cells. Then, the protein samples were separated by 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Billerica, MA, United States). After that, the membranes were incubated with anti-CyclinD1 (1:2,000, bs-20596R, Bioss), anti-Cleaved-caspase3 (anti-Cleaved-casp3, 1:2,000, bs-20364R, Bioss), anti-SOX4 (1:1,000, bs-11208R, Bioss), or anti-β-actin (1:20,000, bs-0061R, Bioss) at 4°C overnight. Goat Anti-rabbit IgG antibody (1:2,000, bs-0295G, Bioss) was used to incubate with the membranes for 2 h. After that, protein signal was detected by BeyoECL Plus reagent (Beyotime).

Total proteins were extracted using radioimmunoprecipitation assay buffer (RIPA) with 1% PMSF (Solarbio, Beijing, China) after transfection for 48 h or induction for six days after transfection. The extracted protein was mixed with protein loading buffer (denaturation) at a ratio of 4:1 and denatured at 100 °C for 10 min. Then, protein was subjected to 10% polyacrylamide gel electrophoresis at 80 V for 30 min and 120 V for 90 min and transferred to a 150 V ice bath for 40 min. The skim milk powder was blocked for 1 h, and the primary antibody was incubated overnight. The antibodies were then rinsed with PBS-Tween three times for 5 min each. The fluorescent secondary antibody was incubated in the dark for 2 h and washed with PBST three times for 5 min each. The primary antibodies used were against cyclin D1 (1:1000, bs-20596R, Bioss), proliferating cell nuclear antigen (PCNA; 1:1000, bs-0754R), peroxisome proliferator-activated receptor γ (PPARγ; 1:1000, bs-4590R), CCAAT/enhancer-binding protein alpha (C/EBPα; 1:1000, bs-1630R), phospho-beta-arrestin (ARRB1; 1:1000, bs-3048R), and GAPDH (1:5000, bs-2188R). The secondary antibody used was goat anti-rabbit IgG H&L/AP (1:5000, bs-0295G-AP). Finally, we present the results of Western blotting using Image Studio Lite ver. 5.2 (LI-COR Inc., Lincoln, NE, USA) and quantified with the ImageJ program (Bio-Rad, Hercules, CA, USA).