RPE sheets cultured on lens capsule for 8 days were microdissected under a dissecting microscope to separate the central region with the heavily pigmented RPE cells, where cell-cell contacts were maintained, from the less pigmented migratory cells at the edges of the sheets. Isolated cells were snap frozen and kept in −80°C until use. Frozen cells were lysed in ice-cold RIPA buffer composed of 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktail and phosphatase inhibitor cocktail I and II (Sigma Aldrich, St. Louis, MO), and protein concentration was determination by BCA assay (Pierce, Rockford, IL). For Western blot analysis, proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked with blocking buffer (SEA BLOCK; Pierce, Rockford, IL) for 1 hour at room temperature, followed by overnight incubation in primary antibodies (one raised in mouse and the other raised in rabbit) in a 1:1 mixture of blocking buffer and PBS at 4°C. Nitrocellulose membranes were washed three times in Tween-20 Tris-buffered saline composed of 30 mM Tris, 150 mM NaCl, and 0.1% Tween-20, pH 7.4, and were incubated for 1 hour with anti–rabbit secondary antibody conjugated with dye (IRDye 800; Rockland Immunochemicals, Gilbertsville, PA) and anti–mouse secondary antibody conjugated with Alexa Fluor 680 dye (Invitrogen). After three washes in Tween-20 Tris-buffered saline and two washes in PBS, membranes were scanned with an infrared scanner (Odyssey; LICOR, Lincoln, NE) to detect and quantify bands.