Retinal Protein Expression Analysis

The neural retina was detached from the RPE, and both tissues were homogenized as previously described [13 (link), 14 (link), 34 (link)]. Proteins (50 μg/sample) were separated in SDS, 12% polyacrylamide gel, transferred to polyvinylidene difluoride membranes, and incubated overnight at 4 °C with a mouse monoclonal anti-cytochrome c (1:1000, Santa Cruz Biotechnology, Dallas TX, USA), a rabbit polyclonal anti-TOM20 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), a rabbit polyclonal anti-VDAC (1:300, Santa Cruz Biotechnology, Dallas, TX, USA), a rabbit polyclonal anti-BDNF (1:500, Alomone Labs, Jerusalem, Israel), a mouse polyclonal anti-RPE65 (1:500; EMD Millipore, Darmstadt, Germany, MAB5428) and a mouse anti-β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). The following secondary antibodies were used: a donkey anti-mouse (1:2000, Jackson Laboratory, Bar Harbor, ME, USA) and a donkey anti-rabbit (1:2000, Jackson Laboratory, Bar Harbor, ME, USA). Densitometric signals were quantified using ImageQuant software and adjusted by the density of β-actin. For each group, the mean of five homogenates were averaged and taken as the representative value.