FIGURE 7.

Epithelial cell transforming sequence 2 (ECT2) binds to and stabilizes focal adhesion kinase (FAK) and is associated with focal adhesion assembly. A, B, Interaction of endogenously expressed ECT2 with FAK examined using total cell lysates from Calu‐3 (A) and NCI‐H2342 (B) cells. A549 (C) and PC‐9 (D) cells were transfected with full‐length Flag‐ECT2 or full‐length Myc‐FAK, then immunoprecipitated with anti‐Flag or anti‐Myc antibodies, and analyzed by Western blotting for Myc, Flag, ECT2, and FAK. IgG was used as a negative control. E, The half‐life of FAK in Calu‐3 cells was assessed by Western blotting after transfection with siECT2#1. The cells were treated with cycloheximide (CHX; a protein synthesis inhibitor) for the indicated times. F, Equal amounts of whole‐cell lysate from Calu‐3 cells transfected with siECT2#1 or siCON for 48 h were immunoprecipitated with anti‐FAK antibody and analyzed by Western blotting for Pho‐Src‐Tyr416, Src, Phos‐Paxillin‐Tyr118, paxillin, Phos‐integrin β1 (Thr788/789), integrin β1, ECT2, FAK, and β‐actin. G, NCI‐H2342 cells were transfected with siECT2#1 or siCON for 48 h. Fixed cells were stained with Phos‐paxillin‐Tyr118 antibody and DAPI. Phos‐paxillin‐Tyr118‐positive focal adhesions were quantified from 20 cells in four pictures using ImageJ software. Error bars represent mean ± SD, scale bar 25 μm, **P < .01, ***P < .001