RGC Protein Extraction and Western Blot

Cell protein extraction and Western blot analysis were performed as previously reported (Wu J. H. et al., 2015 (link); Gao et al., 2017 (link)). Briefly, cultured RGCs were lysed, and total proteins were extracted on ice with cell lysis buffer (Cell Signaling Technology, Boston, MA, USA) and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and then transferred to 0.22-μm PVDF membranes. After blocking with 5% non-fat milk for 1 h, the membranes were incubated overnight at 4°C with primary antibodies against B cell lymphoma 2 (Bcl-2, Abcam, Cambridge, MA, USA), cleaved caspase-3 antibody (Abcam, Cambridge, MA, USA) and β-actin (Abcam, Cambridge, MA, USA). Signals were monitored by the Kodak Imaging System (Kodak 440CF) using ECL Western blot substrate (Hyperfilm ECL, Thermo Fisher Scientific, Rockford, IL, USA). Then, they were quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).