Ba0315

Cells were lysed and the proteins were separated in 10% SDS-PAGE, then transferred to a polyvinylidene difluoride membrane. After blocked with 5% non-fat milk the membranes were incubated with the following primary antibodies at 4°C overnight. Anti- Siva 1 antibody (sc-48767, Santa Cruz Biotechnology), anti- cleaved caspase-3 polyclonal antibody (ab2302, Abcam, Cambridge, UK), anti-Bax antibody (BA0315, Boster, Hubei, Wuhan, China), anti-Bcl-2 polyclonal antibody (BA0412, Boster, Hubei, Wuhan, China) were used. After washing with TBST 4 times for 5 min, the membranes were incubated with anti-rabbit or anti-mouse or anti-goat HRP-conjugated secondary antibody. The protein bands were visualized with a ECL reagent (Wanleibio Co., Ltd.) by the ImageQuant LAS 4000 system (GE Healthcare Life Sciences, Logan, UT, USA). β-Actin (A5316, Sigma) was used for equal loading.

The retinal tissues were lysed on the ice, followed by protein extraction. The protein concentration was determined by BCA assay kit (Wanleibio, Shenyang, China). The proteins were subjected to SDS-PAGE (8–13%) and transferred to PVDF membranes. Then, membranes were blocked with non-fat milk dissolved in Tween-20/TBS buffer. Subsequently, the membranes were incubated with primary antibodies against NgR (1:10,000; ab184556; Abcam, Cambridge, UK), RhoA (1:200; sc-197; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Rock1 (1:200; sc-374388; Santa Cruz Biotechnology, Inc.), Bcl-2 (1:400; BA0412; Boster, Wuhan, China), Bax (1:400; BA0315; Boster), Caspase-3 (1:1000; ab2302; Abcam), F-actin (1:500; ab205; Abcam), growth-associated protein-43 (GAP-43) (1:200; sc-33705; Santa Cruz Biotechnology, Inc.) at 4°C overnight and then with secondary antibody (1:5,000; horseradish peroxidase-labeled IgG; WLA023 and WLA024; Wanleibio) for 45 min at 37°C. The protein bands were visualized via ECL reagent and analyzed by Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).