Ecl western blotting substrate

The transferred blots were blocked with 5% BSA in 0.01% Tween-20 in PBS (PBS-T) with rocking for 1 h at room temperature and then washed 5 times in PBS-T. The blots were incubated with rocking overnight at 4°C with the following primary rabbit monoclonal antibodies in PBS-T with 5% BSA: Anti-Bcl-2 (1:1,000; cat. no. ab32124), anti-Bax (1:1,000; cat. no. ab32503), or anti-Beclin 1 (1:2,000; cat. no. ab207612) (all from Abcam). Following the incubation period, the blots were washed 5 times in PBS-T and then probed with goat anti-rabbit IgG (H+L) horseradish peroxidase secondary antibody in PBS-T with 5% BSA (1:3,000; cat. no. 170-6515; Bio-Rad Laboratories, Inc.) for 1 h at room temperature and then washed 5 times in PBS-T. β-actin was used as a housekeeping protein control and detected using anti-β-actin mouse monoclonal (1:5,000 in 5% BSA; cat. no. ab8226; Abcam) followed by goat anti-mouse IgG (H+L) horseradish peroxidase-labelled secondary antibody (1:3,000 in 5% BSA; cat. no. 170-6516; Bio-Rad Laboratories, Inc.). The blots were then incubated with ECL (Pierce™ ECL Western Blotting Substrate) and the proteins visualised using ODYSSEY^® FC imager (Li-Cor, Bioscience). A semi-quantitative comparison of protein levels was determined by densitometry using Image Studio™ Lite software.