CD317 knockdown leads to activation of Rac. (A) Alexa Fluor 594–phalloidin decoration of F-actin in nonpolarized CD317 knockdown Caco-2 cells transiently expressing either dominant-negative myc-tagged Rac or dominant-negative myc-tagged cdc42 as indicated (x = transfected cells) or after incubation of cells with the ROCK inhibitor Y-27632 as indicated. (B) Immunoblot analysis of phosphorylated Ser19 of MLC (MLCP) in lysates from CD317 knockdown, CD317 rescue, and control Caco-2 cells. An immunoblot of α-tubulin was used as a loading control. (C) Results of pull-down assay for active Rac showing an immunoblot of Rac detected with an anti-Rac antibody. The left lane of each pair shows the total amount of Rac present in 10% of the lysate used in the assay. The right lane of each pair shows the active Rac isolated in the pull-down in each case. Lysates were from the indicated polarized Caco-2 cells. Lysates from PMA-treated cells were used as a positive control for the presence of active Rac. (D) Ezrin and F-actin localization in polarized control (top) and CD317 knockdown (bottom) Caco-2 cells. The left pair of images shows XY sections of the apical surface of the cell monolayer, and the remaining images represent XZ sections of the monolayer. The basal F-actin image (bottom left) shows an XY section of F-actin in the basal region of the polarized CD317 knockdown Caco-2 cells. (E) Immunoblot analysis of C-terminal Thr phosphorylation of ezrin in the indicated Caco-2 cell lysates. Immunoblot of total ezrin was used as a loading control. Black lines indicate that intervening lanes have been spliced out. (B, C, and E) Molecular mass is indicated in kilodaltons. Bars, 10 µm.