Immunobilon p transfer membranes

Retinas were dissected from eye cups, homogenized with lysis buffer (2% SDS in PBS with cocktail protease inhibitor, Sigma–Aldrich Chemie GmbH, Switzerland), and centrifuged at maximum speed (14,000 rpm) for 20 min at 4 °C. Protein concentrations were determined using Pierce BCA Protein Assay kit (Thermo Scientific, Rockford, USA). Protein samples (15 μg per lane) were separated by SDS-PAGE and transferred to Immunobilon-P transfer membranes (Millipore, Billerica MA, USA). The membranes were blocked with Tris-buffered saline (TBS) with 0.1% Tween-20 and 5% skim milk and incubated with anti-RhoA antibody (Santa Cruz, CA, USA) or anti-GAPDH antibody (ZSGB-BIO, Beijing, China), and then with peroxidase-labeled anti-rabbit IgG antibody or anti-mouse IgG antibody (ZSGB-BIO, Beijing, China). The immune complex was visualized under a FluorChem Chemiluminescent Imaging System (Alpha Innotech, San Leandro, CA, USA).