Goat anti rabbit irdye680rd secondary antibodies

RPE cells were cultured for 14 days and lysed by RIPA buffer (Beyotime, Shanghai, China) for 30 min. After centrifugation, total protein concentrations of all samples were quantified using a BCA Protein Quantification Kit (Vazyme) according to the manufacturer’s protocols. Equal amounts (30 μg) of proteins per lane were loaded and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then transferred to nitrocellulose membranes. After blocking with PBS containing 3% BSA for 1 h at room temperature, the membranes were incubated with primary antibodies, including rabbit anti-PRPF6 (1:1000, Invitrogen), rabbit anti-CRALBP (1:1000, Proteintech), mouse anti-RPE65 (1:1000, Abcam), rabbit anti-tyrosinase (1:1000, Abcam), mouse/rabbit anti-GAPDH (1:2000, Arigo) and mouse anti-β-Actin (1:2000, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Then, membranes were incubated with goat anti-rabbit IRDye 680RD secondary antibodies or goat anti-mouse IRDye 800 CW secondary antibodies (1:10,000, LI-COR Bioscience, Lincoln, NE, USA) for 1 h at room temperature. The bands were imaged on the Odyssey Fc Imaging System (LI-COR Bioscience), and the results were analyzed by Fiji/Image J software (National Institutes of Health, Bethesda, MD, USA).