Sc 100 468

Mouse eye tissues and cultured cell pellets were sonicated in RIPA buffer with 1x Halt protease inhibitor cocktail (Thermo Scientific). Lysates of total cellular proteins were collected by centrifugation at 21,000 × g for 15 min at 4°C. The total protein concentration of eye tissue and cell lysates were determined using Bradford Reagent^{55 (link)} or Pierce BCA Protein Assay Kit (Thermo Scientific). The equal amount of protein (20 or 40 μg) was resolved in 10% SDS-PAGE and transferred onto the nitrocellulose membrane. Western blot analysis was performed with primary antibodies: anti-PNPLA2 (1:1000, 2183S, Cell Signaling Technology), anti-CGI-58 (1:2000, sc-100468, Santa Cruz Biotechnology, for Western blot; 1:2000, ab183739, Abcam, for immunostaining), anti-RPE65 (1:1000, house-made antibody),^{49 (link)} anti-LAMP1 (1:1000, 21997-1-AP, Proteintech), anti-Calnexin (1:1000, ab10286, Abcam), Anti-PLIN2 (1:1000, ab108323, Abcam), Anti-PLIN3 (1:1000, 106941-1-AP, Proteintech), and anti-β-Actin (C4) HRP (1:1000, sc-47778 HRP, Santa Cruz Biotechnology). HRP-conjugated secondary antibodies, anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG (1:5000, Vector Laboratories) were used. Western blot signals were acquired using a ChemiDoc system (Bio-Rad), and the signal intensities were quantified using a densitometry application, Image Lab software version 6.1.0 (Bio-Rad).