Cell Lysis and Protein Analysis

For protein analysis, cleared NP40 lysates and whole cell SDS lysates were prepared from 10-cm dishes containing cells at 100% confluency as described [17 (link), 26 (link)]. Protein concentration was measured by BCA assay and 50 micrograms protein was loaded per lane. Immunoprecipitations were performed as described except that the buffer contained 0.5% NP40 [11 (link)]. Antibodies for E-cadherin, N-cadherin, beta catenin, phospho-beta catenin, beta four integrin, and fibronectin were all rabbit polyclonal from Cell Signaling; for Myc-tag (clone 9E10.3), from Neomarkers; for FLAG tag (clone M2), Agilent; for actin (125-ACT), from PhosphoSolutions; for GFP, from Abcam; for vimentin, from Millipore. ZO-1 antibody was from PTG. The CLCA2 antibody TVE20 has been described (12). The protein size marker was Dual color (Bio-Rad). Secondary antibodies were labeled with IR680 or IR800 (Licor), and protein expression was quantified on an Odyssey instrument (Licor).