Versa dostm 4000 mp

Briefly, extracted protein samples from cells were separated by SDS-PAGE, then transferred to nitrocellulose membrane and incubated with the primary antibodies. After incubation with horseradish-peroxidase-conjugated (HPR) secondary antibody for 2 h at room temperature, the protein expression was visualized with BeyoECL Moon Kit (Beyotime, Beijing, China) by Versa DosTM 4000 MP (Bio-Rad Laboratories, Munich, Germany).
Rabbit Anti-SOAT2 antibody (bs-5020R, Biosis, Beijing, China), Rabbit Anti-SREBP2 antibody (bs-2536R, Biosis, Beijing, China), LDL Receptor Rabbit Monoclonal Antibody (AF1438, Beyotime Biotechnology, Shanghai, China), PPAR-γ (I106) polyclonal antibody (BS1587, Bioworld Technology, Inc., MN, USA), CEBP Alpha Rabbit pAb (383901, ZENBIO, Chengdu, Sichuan Province, China), Anti-GAPDH [6C5]—Loading Control (ab8245, Abcam, Cambridge, UK).

The cellular total protein was extracted via addition of RIPA lysis buffer (Beyotime, Shanghai, China) to the cell pellets on ice for 30 min, and protein concentration was measured with a BCA protein quantification kit (Beyotime, Shanghai, China). The protein samples (50 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes (PVDF) and blotted with specific primary antibodies for IGF-1R (Santa Cruz Biotech Inc., sc-713, 1:200 dilution), IRS-1 (Santa Cruz Biotech Inc, sc-560, 1:200 dilution), p-IRS-1 (Tyr 632) (Santa Cruz Biotech Inc., sc-17196, 1:200 dilution ), and β-actin (Santa Cruz Biotech Inc., sc-47778, 1:1000 dilution). After incubation with horseradish-peroxidase-conjugated (HPR) secondary antibody for 2 h at room temperature, the protein expression was visualized with an enhanced chemiluminescent reagent (ECL, Thermo Fisher Scientific, City, MA, USA) by Versa DosTM 4000 MP (Bio-Rad Laboratories, Munich, Germany).