Ha tag

Cells were lysed at 4°C in RIPA buffer (50 mM Tris, pH 8, 1% Triton, 150 mM NaCl, 0.5% SDS, 50 mM Triton, 1 mM EDTA) + Protease Inhibitor Cocktail (Roche, 11697498001), sonicated for 15 s, clarified via centrifugation, and stored in –80°C. Lysates were solubilized by mixing 1:1 in loading buffer (10% SDS, 40% glycerol, 3% Bromophenyl Blue, and 10% Beta-Mercaptoethanol). Proteins in lysates with loading buffer were first denatured by boiling for 5 min and cooled on ice for 2 min before being loaded into 10% polyacrylamide gels and run for ∼90 min at 125V. Resolved proteins were transferred onto nitrocellulose membranes for 1 h at 100V. Membranes were then blocked with 5% bovine serum albumin (BSA) for an hour before incubation with primary antibody, washed three times in PBST (2.0% Triton in phosphate-buffered saline [PBS]), and then finally incubated with secondary antibodies (Licor, IRDye 680RD Series, CW800 Series). Bands were visualized using a LI-COR Odyssey FC system. Primary antibodies used were Dsg1 (BD Bioscience, 610273), Desmoplakin (Millipore Sigma, MABT1492), E-cadherin (Invitrogen, 13-1900), GST (Bethyl Laboratories, A190-122P), HA tag (Roche, 11867423001), Occludin (Thermo Fisher, 71-1500), Plakoglobin (Santa Cruz, sc-7900), and Streptavidin-HRP (Thermo Fisher, 43-4323).