Loss of ezrin/radixin or LOK/SLK in DLD-1 colon epithelial cells. (A) Protein expression using the indicated antibodies in Western blots of DLD-1 mixed knockout cell lysates. In DLD-1 LOK^−/− SLK^−/− cells, pERM is undetected. HeLa cell lysate was used as a control for expression of moesin, which is not present in DLD-1 cells. Tubulin expression was used as a loading control. (B) DLD-1 wild-type and mixed knockout populations of Ezr^−/− Rdx^−/− cells and LOK^−/− SLK^−/− cells stained with ezrin, ZO-1, and actin at 40× magnification. (C) DLD-1 wild-type and mixed knockout populations of Ezr^−/− Rdx^−/− cells and LOK^−/− SLK^−/− cells stained with ezrin and actin at 100× magnification. Vertical projections were expanded fourfold for clarity. Magnified insets highlight major differences between actin organization in wild-type and knockout cells. (D) Representative Western blot of active RhoA-GTP pull-down results in DLD-1 cells. Upper row: RhoA blot of the active fraction of RhoA-GTP pulled down by rhotekin beads. Lower row: Blot of RhoA of 5% of input lysate. (E) Quantification of pull-down represented in D. Mean ± SEM; n = 6 (t test; *, P ≤ 0.05). Scale bars, 10 µm.