Fig. 5. The effect of Foxj1 on actin assembly and RhoA activation. (A) Confocal photomicrographs of actin (phalloidin, red) and basal body (-tubulin, green) localization in mTEC cultures (ALI longer than day 7) from wild-type (WT) or Foxj1^–/– mice. Reconstructed confocal z-axis images are below. (B) 3D reconstruction of confocal microscopy images from A showing the loss of apical actin in Foxj1^–/– compared to WT mTECs. (C) Western blot showing RhoA and RhoB GTPase activation in non-differentiated mTECs infected with AdGFP or AdFoxj1. GTP-bound RhoA and RhoB were isolated from cell lysates by binding to rhotektin-agarose, and subjected to immunoblot analysis. RhoA and RhoB were detected in the same membrane re-probed with specific antibodies. (D) Scanning densitometry of immunoblots from C showing active relative to total Rho. (E) Western blot showing Rac1 GTPase activation in mTEC cultured as in C, treated with hepatocyte growth factor (HGF, 50 ng/ml, 4 hours) or transfected with adenovirus vector expressing GFP or Foxj1. Rac1-GTP was isolated from cell lysates of undifferentiated mTECs by binding to P21-binding-domain-bound agarose and immunoblots analyzed. (F) Scanning densitometry of blots shown in E.