HPAEC were transfected with non-specific, Tiam1/Vav2-, or Rac-specific siRNAs for 48 hr, followed by OxPAPC stimulation (10 μg/ml, 15 min). A: Cytosolic and membrane fractions were separated according to the protocol described in Methods, and translocation of p190RhoGAP to the membrane fraction was detected with specific antibodies. Inset - western blot analysis of Tiam1 and Vav2 depletion in HPAEC. B: After cell lysis step, protein complexes were immunoprecipitated with p120-catenin antibodies followed by western blot analysis with p190RhoGAP antibody. Equal protein loading was confirmed by reprobing of membranes with p120-catenin antibody. C: p190RhoGAP was immunoprecepitated under denaturing conditions. Phosphorylation status of p190RhoGAP was determined by western blot analysis with p-tyrosine antibody. Equal protein loading was confirmed by probing of membranes with p190RhoGAP antibody. Rac depletion was confirmed by probing of membranes with specific antibody. Result of densitometry shown as mean ± SD, * p<0.05