Sirt3 c73e3 rabbit mab

Proteins were extracted from cells and tissues using RIPA lysis buffer (Biosharp, Shanghai, China). Protein concentrations were determined by a BCA protein assay kit (NCM Biotech, Suzhou, China). All cell lysates containing 40 μg of protein were subjected to SDS-PAGE and electrophoretically imprinted on PVDF membranes. The membrane was blocked with Tween-Tris buffered brine (TTBS) containing 5% skim milk at room temperature for 2 h and then incubated with the following primary antibodies: SIRT3 (C73E3) rabbit mAb (2627, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-LCAD antibody (2980, Cell Signaling Technology), rabbit anti-GDH antibody (12793, Cell Signaling Technology), rabbit anti-ACE2 antibody (92485, Cell Signaling Technology), and GAPDH (4970, Cell Signaling Technology). The dilution of primary antibodies was 1:500. After that, the membrane containing protein bands was incubated with HRP polymerized secondary antibody (1:500, bs-0296G-HRP) at room temperature for 2 h. The bands were visualized by an ECL chemiluminescence detection system, and the protein expression was analyzed by ImageJ software for optical density values. Triplicates were performed for each sample to ensure accuracy and reproducibility of the results.