Because of limited sample availability, Western blot analysis was performed at two ages (7 and 16 weeks), with one normal and one mutant retina used at each time point. Protein extraction and Western blot analysis were performed as described elsewhere, with minor modifications.
39 Briefly, normal and mutant retinas were homogenized and sonicated at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The homogenate was centrifuged at 13,000
g for 15 minutes at 4°C, and the supernatant containing the proteins was collected. Total protein levels were determined by the Bradford method (ABC protein assay; Bio-Rad, Hercules, CA). For Western blot analysis, 20 μg protein extract was boiled in SDS sample buffer (4% glycerol, 0.4% sodium dodecyl sulfate, 1% β-mercaptoethanol, and 0.005% bromophenol blue in 12.5 mM Tris-HCl buffer [pH 6.8]) and then separated, along with a biotinylated protein ladder (no. 7727, 1:1000; Cell Signaling Technology, Danvers, MA), by SDS-PAGE (4% stacking gel, 12% separating gel). The proteins were transferred to a polyvinylidene difluoride membrane (Trans-Blot Transfer Medium; Bio-Rad) in chilled transfer buffer (25 mM Tris base, 192 mM glycine, and 15% methanol). The membrane was blocked in 10% skim milk in Tris-buffered saline containing 0.5% Tween-20 overnight at 4°C and then was incubated for 1.5 hours with the primary antibodies. The antibodies included were NDUFS4 (ab55540, 1:1,000; Abcam, Cambridge, MA), SAG
40 (kindly provided by Igal Gery, 1:10,000), GFAP
27 (Z0334, 1:10,000; Dako Cytomation, Carpinteria, CA), and OPN1SW
27 (AB5407, 1:2,000; Chemicon, Temecula, CA). With the exception of NDUFS4 (specificity determined by the detection of the appropriate size product by Western blot analysis), the other antibodies had been validated and produced specific labeling in the canine retina using Western analysis and/or immunohistochemistry
27,40 (Kathleen Boesze-Battaglia, School of Dental Medicine, University of Pennsylvania, Philadelphia, personal communication, 2010). ACTB (MAB1501, 1:10,000, Chemicon) was used as the loading control.