Trans blot turbo pvdf transfer pack and system

IPSC-derived RPE grown in 24-well plates was scraped in cold PBS containing Complete protease inhibitor cocktail tablets (Roche) and centrifuged at 3000g for 5 min. The pellet was resuspended in 2× Laemmli's sample buffer (Biorad) containing 1/25 dilution of β-mercaptoethanol (Sigma-Aldrich) and Benzonase (Sigma-Aldrich). The samples were heated 5 min at 95 °C and loaded onto an AnyKD precast MiniProtean TGX Stain Free gel (Biorad). The separated proteins were electrotransferred using a Trans-Blot® Turbo™ PVDF Transfer Pack and System (Biorad). After blocking for 1 h in 0.5% Tween-PBS in 5% skim milk (blocking solution), membranes were incubated with 1:250 dilution of anti-ZO-1, 1:500, dilution of anti-N-Cadherin, 1:2000 dilution of anti-β-catenin, 1:2000 dilution of anti-MERTK, 1:1000 dilution of anti-CRALBP or with 1:1000 dilution of anti-GAPDH overnight at 4 °C. After 3 washes in 0.5% Tween-PBS, the membrane was incubated with 1:10,000 dilution of horseradish peroxidase (HRP)-conjugated sheep antibody against mouse (Jackson ImmunoResearch, Interchim, Montluçon), or goat antibody against rabbit (Sigma-Aldrich), whole immunoglobulins. The detection step was performed using the Amersham ECL prime western blotting detection reagent (GE Healthcare) and a Biorad Chemidoc XRS+ system.