N cadherin sc 271386

Cells were lysed in RIPA Buffer (50 mM Tris Base, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.25% sodium deoxycholate) supplemented with protease and phosphatase inhibitors (Halt™ Thermo Scientific). Equal protein concentrations of total cell lysates, as determined by the Coomassie Plus Protein Assay (Thermo Scientific), were separated by SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membranes (BioExpress). Membranes were blocked in 5% non-fat milk in TBST (1.0 M Tris-HCL, 5.0 M NaCl, 0.1% Tween) for 1 hour at room temperature, then incubated with primary antibody (LSR, SC133765; TIAM1 SC827; FLT1 SC316, N-Cadherin SC271386, Santa Cruz Biotechnology; AF6 610732, BD Transduciton Labs; Claudin7 34–1500, Occludin 33–1500, Zymed; PKCζ CST9372, Cell Signaling) overnight at 4°C in TBST containing 5.0% BSA, washed, and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (GE Healthcare) in TBST with 5% milk for 1 hour at room temperature. Mouse monoclonal α-tubulin antibody was used as a loading control at 1∶5000 dilution (T6199; Sigma Aldrich). Enhanced chemiluminescence detection system (ECL Plus, GE healthcare) was used to detect peroxidase activity. NIH Image J64 was used to quantify western blots.