Horseradish peroxidase conjugated secondary antisera

For Western blotting, ARPE19 cells were seeded at a density of 2.5 × 10⁵/well and treated with the indicated compounds. For pRPE cell analysis, cells were cultured until confluence and fully pigmented (∼45 days). After indicated treatments, cells were lysed in RIPA buffer with protease inhibitors (Santa Cruz, Santa Cruz, CA) and protein quantification was performed using the bicinchoninic acid protein assay (Thermo Scientific, Waltham, MA). Protein samples (10 μg) were resolved through 4%–12% gradient acrylamide sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (NuPage Novex; Life Technologies). Western blots were blocked in 5% nonfat milk in Tris-buffered saline/Tween 20 and observed using anti-β-catenin, α-SMA, RPE65, microphthalmia-associated transcription factor (MITF; Abcam), acetylated α-tubulin, and α-tubulin (Cell Signaling, Bedford, MA) followed by the appropriate horseradish peroxidase-conjugated secondary antisera and enhanced chemiluminescence detection following the manufacturer's instructions (Bio-Rad, Hercules, CA).