Las 4000 mini luminescence image analyzer

Retina lysates from Atxn3+/+ and Atxn3−/− mice were obtained by homogenization and sonication in RIPA lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP40, with protease inhibitors (Complete, Roche Diagnostics, Indianapolis, IN)). Proteins from Atxn3+/+ and Atxn3−/− mouse RPE were extracted following a previously described protocol (Wei et al., 2016 (link)). Proteins (30 μg) were resolved in 10%–12.5% SDS-PAGE gels, transferred onto PVDF membranes and blocked with 5%–10% non-fat milk in PBS-T for 1 h, followed by an overnight incubation at 4°C with primary antibodies and a peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:2000) for 1 h. Bands were visualized by treatment either with the ECL-plus reagent (Western Lightning®, PerkinElmer, Waltham, MA) and exposure to autoradiography films or with Lumi-Light Western Blotting Substrate (Roche Diagnostics, Indianapolis, IN) and chemiluminescence detection in a LAS-4000 mini Luminescence Image Analyzer (Fujifilm, Tokyo, Japan). Fiji software (Schindelin et al., 2012 (link)) was used to quantify protein band density and α-tubulin or GAPDH were used for normalization. Figure 4 shows representative western blots (n = 4). Uncropped blots are shown in Figure S5D.