Goat anti rabbit and anti mouse hrp conjugated secondary antibodies

Whole retinas were lysed in RIPA buffer containing protease inhibitors (Thermo Fisher Scientific). Supernatants were cleared by centrifugation at 12,000g for 15 minutes (4°C), and protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific). Protein samples (30 μg) were separated on 10% SDS polyacrylamide gels, transferred to PVDF membranes, and probed with polyclonal rabbit antibodies raised against TRPV2 (1:500; Merck; PC421) and mouse monoclonal anti–β-actin antibodies (1:5,000; Cell Signaling Technology; 8H10D10). After washing, membranes were incubated with appropriate goat anti–rabbit and anti–mouse HRP-conjugated secondary antibodies (1:2,000; Cell Signaling Technology; catalogs 7074 and 7076S). Protein bands were visualized by enhanced chemiluminescence and imaged using a Syngene G:BOX Chemi XX6system (Syngene). Immunoblots were quantified by densitometry, and TRPV2 protein expression was normalized to β-actin levels.