After appropriate treatments and rinsing with cold PBS, REC or whole retinal lysates were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein from the cell extracts were separated on the precast 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 membranes was treated with Epac1, PKCzeta, Occludin, ZO-1, VEGF (all from Abcam, San Francisco, CA, USA), or beta actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA, USA). Data was acquired using an Azure C500 (Azure Biosystems, Dublin, CA, USA). Western blot analyses were done using Image Studio Light software (LI-COR, Lincoln, NE, USA).