Western blots

The induced photoreceptor-directed fibroblasts were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM tris pH 8.0, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate and 1% NP40 substitute) containing 1X protease inhibitor cocktail (Roche). The cell lysates were homogenized with gentle agitation for 30 min on ice and centrifuged at 12,000 rpm at 4 °C for 20 min. Each supernatant was collected in a new microcentrifuge tube. The protein concentration of each sample was measured by the Pierce™ BCA Protein Assay Kit (ThermoScientific). The samples were denatured with 4 X Laemlli sample buffer (Biorad), heated at 95 °C for 5 min, resolved in 4–20% gel (Biorad) and transferred to PDVF membrane (Biorad). Detection of immune complexes formed by proteins of interest and primary antibodies (anti-blue opsin (1:250, Milipore, AB5407), anti-CHOP (1:1500, Cell Signaling Technology, #2895), anti-cleaved caspase-3 (1:1000, Cell signaling technology, #9664), anti-β-actin (1:1000, Cell Signaling Technology)) was performed by enzyme-linked color development with anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000, Cell Signaling Technology). The signals based on chemical luminescence (ECL™ Prime Western Blotting system (Amersham)) were detected by ChemiStage (TOYOBO). The band intensity was quantified with ImageJ software (National Institutes of Health).