Anti phospho pkcζ

Whole retinal lysates and REC lysates were placed into lysis buffer containing protease and phosphatase inhibitors. The lysates were kept on ice for 30 min and cleared by centrifugation at 12,000 rpm for 20 min at 4 °C. Equal amounts of protein from the lysates were separated on the precast tris-glycine gels (Invitrogen, Carlsbad, CA) and blotted onto a nitrocellulose membrane. The blots were blocked with TBST (10mM Tris-HCl buffer, pH 8.0, 150mM NaCl, 0.1% Tween 20) containing 5% (w/v) bovine serum albumin and then incubated with respective primary antibodies. Primary antibodies used were anti-phospho-PKCζ, PKCζ, PKCδ, phospho-PKCδ, phospho-eNOS and eNOS (Cell Signaling, Danvers, MA). IGFBP-3, VEGF and β-actin antibodies were purchased from Santa Cruz Technology (Santa Cruz, CA). Membranes were then washed with TBST and incubated with horseradish peroxidase labeled secondary antibodies. The antigen–antibody complexes were detected using West Pico Chemilluminescence reagent. Mean densitometry of the bands were assessed using Kodak Image Station 4000MM software (Carestream, Rochester, NY).

The protein concentrations of samples were measured using bicinchoninic acid (BCA) protein assay (Thermo Scientific, 23228). Proteins (~30 μg) were mixed with 1× NuPAGE LDS Sample Buffer (ThermoFisher, NP0007) and 1× NuPAGE Sample Reducing Agent (ThermoFisher, NP0009), boiled for 5 min before applying to SDS–polyacrylamide gel electrophoresis. The following are the antibodies we used in this study: anti-Gapdh (Santa Cruz, sc-25778), anti-Akt (Cell Signaling, 9272s), anti–phospho-Akt (Cell Signaling, 9275s), anti-Cers5 (Life Technologies, PA-520570), anti-Cers6 (Santa Cruz, sc-100554), anti–β-actin (Santa Cruz, sc-47778), anti-PKCζ (Santa Cruz, sc-216), anti–phospho-PKCζ (T410, Cell Signaling, 2060S), anti-Ppp2ca (Cell Signaling, 2041S). The intensity of the bands was quantified using ImageJ software (National Institutes of Health) and normalized to Gapdh or β-actin.