Western blot analysis

Isolated retinal tissue including the RPE was incubated in 2 mM CaCl₂ at room temperature for 30 min and then in buffer containing 150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 1% NP-40, complete protease inhibitor cocktail and PMSF (Roche), as previously described⁵². Total proteins (50 µg) were separated by SDS-PAGE and transferred onto PVDF membrane. Membranes were immersed in a solution of 5% non-fat milk and 0.1% Tween 20 in Tris Buffered Saline (TBS) for 2 h and then incubated with the appropriate primary antibodies diluted in blocking solution at 4 °C overnight. The primary antibodies used were: rabbit antiserum against the C-terminal domain of PCDH21 (a kind gift of Dr. A. Rattner; 1:10.000), mouse monoclonal antibody generated against the C-terminal domain of N-cadherin (Invitrogen; 1:500); anti-CRB1 (a kind gift of Dr. J. Wijnholds, 1:500), and mouse anti-α-tubulin (Sigma, 1:10.000), used as a loading control. The membranes were washed and incubated with horseradish peroxidase-conjugated rabbit or mouse antibodies followed by ECL Advanced Western Blotting Detection Kit (Amersham). The immune-reactive bands were quantified by densitometry and the amount of proteolyzed fragment was estimated as the ratio of C-terminal fragment (CTF)/full length (FL)/α-tubulin band values. Western blots were repeated at least 3 times obtaining similar results.