Complete protease inhibitors and phosphatase inhibitors

Following euthanasia, eyes were enucleated and placed in ice-cold PBS (pH 7.4). Retinas were dissected from eyecups and lysed in radioimmunoprecipitation assay buffer (Cell Signaling, Danvers, MD, USA) with complete protease inhibitors and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Protein lysates (20 μg) were run on a 4% to 15% gradient gel by SDS-PAGE and transferred to a polyvinylidene fluoride membrane overnight. Membranes were blocked in 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T, pH 7.4) for 1 hour at room temperature (RT) and incubated with primary antibodies (rabbit anti-Kir4.1, 1:400 in 1% BSA; Millipore Corp., Billerica, MA, USA, and mouse anti-GAPDH, 1:1000 in 2% BSA; Abcam, Cambridge, UK) overnight at 4°C. Following 3 × 15 minute wash with TBS-T, horseradish peroxidase–conjugated secondary antibodies (1:20,000 in 5% milk TBS-T) were incubated for 1 hour at RT. Membranes were washed 4 × 15 minutes with TBS-T then rinsed once in TBS. Proteins were detected by chemiluminescene (Western Lightning ECL Pro; Perkin Elmer, Waltham, MA, USA). Densitometry was performed using ImageJ analysis of scanned films (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).