Rhoa sc 179

Following treatment, monolayers were placed on ice and washed with cold PBS. Cells were lysed using protein lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 30 mM Na₄P₂O₇, 50 mM NaF, 1 mM Na₃VO₄, 1% Triton X-100, 1 mM PMSF, 10 ug/mL leupeptin, 10 ug/mL aprotinin, and 10 ug/mL pepstatin). Lysates were collected using cell scrapers, transferred to microcentrifuge tubes, and centrifuged at 14000 rpm for 15 minutes at 4 °C. Protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s protocol. Supernatant was combined with 6X SDS-buffer, heated at 65 °C for 5 minutes, and stored at −20 °C.
Samples were resolved on SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, and blocked for 1 hour in 5% milk in TBST. Membranes were incubated with primary antibody diluted (1:1000) in antibody buffer overnight. The following day, membranes were washed and incubated with secondary antibody diluted (1:10000) in 5% milk in TBST for 1 hour at room temperature. Membranes were then developed using ECL, film, and radiography.
Primary and secondary antibodies were all from Santa Cruz Biotechnology: EPHA4 (sc-921), Cdc42 (sc-8401), Rac (sc-11), RhoA (sc-179) and β-tubulin (sc-2005). Secondary antibodies were goat anti-rabbit IgG-HRP (sc-2030) and goat anti-mouse IgG-HRP (sc-2031).

Individual dissected posterior eyecups were lysed in 160 μL of HNTG detergent buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl₂, 1% Triton X-100) freshly supplemented with 1% protease and phosphatase inhibitor cocktails (#P8340, Sigma-Millipore, and #78420, Thermofisher, respectively). Cleared lysates representing equal eyecup tissue fractions were separated on 12% SDS polyacrylamide gels using a standard Tris–glycine buffer system (Novex gels and solutions, Thermofisher). Proteins were transferred to nitrocellulose membranes (#88018, Thermofisher) for immunodetection with primary and appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The primary antibodies used were porin (#4866, Cell Signaling, 1:5,000), RhoA (#sc-179, Santa Cruz Biotechnologies, Santa Cruz, CA, USA, 1:200), rhodopsin (clone B6-30, 0.01 μg/mL), and α-tubulin (#9099, Cell Signaling, 1:5,000). The secondary antibodies used were donkey-anti-rabbit IgG-HRP (#16029, Thermofisher, 1:10,000) and donkey-anti-mouse IgG-HRP (#16011, Thermofisher, 1:5,000). Chemiluminescence reaction signals from ECL-plus substrate (#NEL105001EA, Perkin-Elmer, Shelton, CT, USA) were captured with X-ray films (#3018, Denville Scientific, Swedesboro, NJ, USA), which were scanned and quantified by densitometry using Image Quant^TM TL 7.0 (GE Healthcare, Chicago, IL, USA).