Analysis of p190A mutations found in tumors. (A) Schematic representation of the different p190A mutants. Point mutations, deletion, and the PLS domain are indicated on the protein. (B) Huh7 cells transfected with p190AWT or p190A mutants were plated for 3 h on fibronectin, fixed, and stained for HA (green), F-actin (red), and nuclei (blue). NT, nontransfected cells. Arrowheads show localization of mutants at cell protrusions; * shows cytoplasmic localization of mutants; and # points out absence of stress fibers. (C) Localization of p190A mutants is analyzed by quantification of membrane staining intensity/cytoplasmic staining intensity ratio. The graph presents the mean ± SEM of three independent experiments (n = 20 cells per condition). **, P < 0.01; ***, P < 0.001. (D) Quantification of cells bearing stress fibers in the experiment described in B. Statistical significance was calculated relative to the control (p190WT) condition. ***, P < 0.001. (E) Huh7 cells were transfected with indicated constructs or mutants, and their affinity for active RhoA was tested by pulldown using recombinant GST-RhoAQ63L and revealed by immunoblot with anti-HA antibodies. Quantification of the affinity between p190A mutants and active RhoA is represented as a histogram in which values were calculated by measuring the band intensity of pulldown/input and are represented as the mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01.