Western Blot Analysis of Retinal Proteins

Protein preparation and Western blotting were performed according to previously described methods (Zhou et al., 2017a (link)) with modifications. Briefly, retinal lysates were centrifuged at 12,000 rpm for 10 min at 4 °C. In total, 20 μg of each sample was separated by SDS-PAGE and electrotransferred onto PVDF membranes (Immobilon-P; Millipore). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: GABA-alpha receptor (GABAAR; ab33299, rabbit, 1:500; Abcam), protein kinase C-alpha (PKC-α; ab32122, rabbit, 1:1,000; Abcam), and B-cell lymphoma-2 (rabbit, 28 kDa, 1:1,000, Beyotime Institute of Biotechnology, China). The membranes were then incubated with horseradish peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1:5,000; Cell Signaling Technology). The relative intensities of the protein bands were quantified by scanning densitometry using ImageJ software. Mouse monoclonal antibodies against β-tubulin (ab78078, 1:2000; Abcam) and β-actin (mouse, 43 kDa, A5441, 1:5,000, Sigma-Aldrich, USA) were used as internal standards.