To prepare retinal samples for Western blotting, trephined retinae (7-mm diameter buttons) were lysed, homogenized in 1× radioimmunoprecipitation assay buffer (EMD Millipore, Billerica, MA, USA) containing 1× protease inhibitor (Roche, New York, NY, USA), 2 mM Na
3VO
4, and 10 mM NaF with ceria stabilized zirconium oxide beads (0.5 mm in diameter, ZrOB05; Next Advance, Averill Park, NY, USA), and centrifuged at 15,000
g for 10 minutes at 4°C. Protein concentrations were determined using a protein assay kit (Quick Start Bradford protein kit; Bio-Rad, Hercules, CA, USA). The samples were then dispersed in an equal volume of 2× SDS loading buffer (Bio-Rad) containing 10% 2-mercaptoethanol, and boiled for 5 minutes; assayed by electrophoresis, using 10% or 8% to 16% precast polyacrylamide gels (Bio-Rad); transferred to a nitrocellulose membrane (0.2 μm; Bio-Rad), and analyzed by Western blotting. The following antibodies were used: RhoA rabbit monoclonal antibody (mAb), phosphorylated myosin light chain 2 (pMLC [Ser-19]) mouse mAb, myosin light chain 2 (MLC) rabbit mAb and protein kinase C alpha (PKCα) rabbit polyclonal antibody (pAb) (all from Cell Signaling, Boston, MA, USA), di-phosphorylated MLC 2 (di-pMLC [Thr-18/Ser-19]) rabbit mAb (Thermo Scientific, Rockford, IL, USA), RAC1 mouse mAb (Becton Dickinson, Franklin Lakes, NJ, USA), p-CPI-17 rabbit pAb (Santa Cruz Biotechnology, Dallas, TX, USA), synaptic vesicle protein 2 (SV2) and myosin IIB mouse mAb (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and glial fibrillary acidic protein (GFAP) rabbit pAb (Dako, Carpinteria, CA, USA). For an internal control, glyceraldehyde-3-phosphate dehydrogenase rabbit (GAPDH) pAb (Santa Cruz Biotechnology) was blotted in the same membrane. The membranes were visualized using secondary goat anti-mouse or rabbit immunoglobulin G (IgG) antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Luminata Classico or Forte Western horseradish peroxidase substrates (EMD Millipore) were used to visualize the immune complexes. The density of a specific band was determined using ImageJ software (version 1.45s; US National Institutes of Health, Bethesda, MD, USA,
http://imagej.nih.gov/ij/, available in the public domain).