Western Blot Analysis of Autophagy Markers

Western blot was performed by using a standard protocol as previously described [29 (link)]. Samples were normalized to 1 mg/mL and the loading volume was 20 mL/well. The membranes were, respectively, incubated with rabbit polyclonal anti-LC-3B (1 : 500; Novus Biologicals), rabbit polyclonal Beclin-1 (1 : 1000; Abcam Cambridge, UK), rabbit polyclonal ZIP8 (1 : 1000; Santa Cruz Biotechnology), rabbit polyclonal nuclear factor-kappa beta (NF-κβ) (1 : 1000, Abcam Cambridge, UK), rabbit polyclonal anti-iNOS antibody (1 : 1000, Abcam Cambridge, UK), and rabbit anti-β-actin (1 : 500; Santa Cruz Biotechnology). The bound antibodies were detected using goat anti-rabbit IgG-HRP antibody (1 : 1000; Abcam Cambridge, UK). The protein bands were visualized by an ECL detection system (Pierce Chemical, Rockford, IL, USA) and quantified by Image J software (NIH, Bethesda, MD).