After appropriate treatments and rinsing with cold PBS, REC were lysed in the lysis buffer containing the protease and phosphatase inhibitors, and scraped into the tubes. Retinal extracts were prepared by sonication. Equal amounts of protein from the cell or tissue extracts were separated on the pre-cast tris-glycine gel (Invitrogen), blotted onto a nitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0; 150 mM NaCl; 0.1% Tween 20) and 5% (wt/vol) BSA, the membrane was treated with appropriate primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen–antibody complexes were detected by chemiluminescence reagent kit (Thermo Scientific, Waltham, MA). Primary antibodies used were phosphorylated Akt (Serine 473), Akt, Cytochrome C, Bax, Bcl-xL (all purchased from Cell Signaling, Danvers, MA), beta actin (Santa Cruz Biotechnology, Dallas, TX), and IGFBP-3 (GroPep Bioreagents Pty Ltd, Adelaide, Australia). Western blot analyses of proteins of interest were compared to beta actin levels, and a ratio is presented.