Anti gapdh antibody

First, total protein was extracted from MG-63 cells and hFOB1.19 cells using RIPA buffer. Next, complete proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were next closed with 5% skim milk powder in TBST at 37°C for 1h and incubated with primary antibodies at 4°C overnight. The antibodies were anti-INSR antibody (#38126, Signalway Antibody LLC, SAB, USA), anti-GAPDH antibody (#23001, Signalway Antibody LLC, SAB, USA) antibody as an internal reference. Then TBST was washed for 20 minutes and incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody for 1 hour at 37°C. Finally, the protein bands were observed with an enhanced chemiluminescence kit.

Tissues were homogenized in RIPA buffer and then centrifuged at 11,000 × g for 10 min at 4°C. Equal amounts of protein lysates were loaded on an SDS-polyacrylamide gel. Anti-SREBP-2, anti-SREBP-1c, and anti-adiponectin antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-p-AMP-activated protein kinase (AMPK) and anti-p-Acetyl CoA carboxylase (ACC) antibodies from Cell Signaling Technology (Beverly, MA, USA), anti-LDLR antibody from BioVision (Milpitas, CA, USA), and anti-GAPDH antibody from Signalway Antibody (College Park, MD, USA) were used to detect the respective proteins.