Western Blot Analysis of Retinal Vascular Proteins

Western blot analysis was performed as described in our previous study of retinal arterioles with slight modification.^{26 (link),46 (link)} Retinal venules (sample pooled from both eyes per pig) and neural retina tissue were isolated and sonicated in lysis buffer. The protein content of each lysate was determined with the BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein (2.5 μg for ET-1 receptors and 5 μg for ROCK isoforms and p38) were separated by Tris-glycine SDS-PAGE (4%–15% Tris-HCl Ready Gels; Bio-Rad, Hercules, CA, USA), transferred onto a nitrocellulose membrane, and incubated with rabbit anti-ET_AR or anti-ET_BR polyclonal antibody (1:250 dilution; Catalog nos. sc-33535 and sc-33537; Santa Cruz Biotechnology, Santa Cruz, CA, USA),^{26 (link)} or mouse anti-ROCK1 or anti-ROCK2 monoclonal antibody (1:250 dilution; Catalog nos. sc-17794 and sc-398519; Santa Cruz Biotechnology). Membranes were stripped and reprobed with rabbit anti-p38 antibody (1:1000; Catalog no. sc-535; Santa Cruz Biotechnology), which we previously have shown is highly expressed in neural retina tissue.^{26 (link)} After incubation with an appropriate secondary antibody (anti-rabbit or anti-mouse IgG, 1:1000; Catalog nos. 7074S and 7076S; Cell Signaling Technology, Danvers, MA, USA), the membranes were washed and developed by enhanced chemiluminescence (Pierce).