Fig. 2.

RhoA activity is required for Shroom3 induced apical constriction. (A-D) MDCK cells were transfected with the indicated expression plasmid and subsequently treated with the specified chemical inhibitor. Immunofluorescent labeling of ZO1 (green) was used to visualize the apical junctions, and for the epitope tags (red), to visualize transfected RhoA (B) or Shroom3 (D). Apical views of cells are visualized in the xy plane and the apical-basal axis is visualized in the xz plane. The average apical area for cells was calculated for each condition and is displayed in the bar graphs (A,C). Error bars represent s.e.m. ^*P<0.05. (E) The amino acids 594-1060 of Rock1 fused to GST, constitutively active RhoA (RhoA^L63) and the Rho-kinase binding domain of Shroom3 (SD2) were bacterially expressed and used in a pull-down assay. Coomasie Blue staining of an SDS-PAGE gel reveals protein that was resolved from the pellet fraction (P), which indicates binding to GST-Rock1, or from the supernatant (S).