Rabbit anti beclin1 antibody

The proteins were separated on a 6%/12%/15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and then electrotransferred onto a polyvinylidene difluoride membrane (Millipore). The membranes were blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat dry milk for 1 h at RT. Thereafter, different primary antibodies, including rabbit anti-LC3B antibody (1:500; Cell Signaling Technology), rabbit anti-Beclin-1 antibody (1:1,000; Cell Signaling Technology), rabbit anti-p62 antibody (1:1,000, Cell Signaling Technology), rabbit anti-GAPDH antibody (1:10,000; Abcam), and mouse anti-β-tubulin (1:5,000, FDbio Science, Hangzhou, China) were used to incubate the membranes overnight at 4°C. After washing three times, the membranes were incubated by horseradish peroxidase-labeled secondary antibody (1:5,000; Beyotime) for 1 h at RT. Immunoblot was detected with the enhanced chemiluminescence (ECL) substrate (Bio-Rad) and digitized by GelView 6000Pro (BLT, Guangzhou, China). Band densities were quantified using the ImageJ 1.52 software program (National Institutes of Health). The quantities of the band densities were normalized to a loading control GAPDH or β-tubulin and then compared among the three groups.