Sc 374388

Total proteins were isolated from tissues using an active protein extraction kit (KGP1050; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). A BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. A total of 20 µg protein per lane was separated using 10% SDS-PAGE, transferred onto polyvinylidene difluoride membranes and then blocked with 5% fat-free milk at room temperature for 2 h. Membranes were then incubated with primary antibodies detecting ROCK1 (sc-374388; 1:1,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (sc-293335; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following two washes with Tris-buffered saline with 0.5% Tween-20 (TBS-T), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; ZB-2306; OriGene Technologies, Inc., Beijing, China) for 2 h at room temperature and then washed twice with TBS-T. Proteins were detected using enhanced chemiluminescence RapidStep™ ECL, according to the manufacturer's protocol (cat. no. 345818; Merck KGaA). ImageJ (version 1.8.0; National Institutes of Health, Bethesda, MD, USA) was applied to quantify the relative protein levels. GAPDH was used as an internal control. The integral optical density ratio of ROCK1/GAPDH indicated the relative expression of ROCK1 protein.

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).