Fig. 2. Actin cytoskeleton configuration regulates p190RhoGAP localization. (A) Levels of active RhoA in p190RhoGAP knockdown HMVE cells in the presence or absence of cytoD 1 hour after replating on fibronectin-coated plates. Quantitative results were normalized to spread control cells without cyto D (^*P<0.01). (B) Immunoblots showing lipid raft localization of p190RhoGAP in spread versus cytoD-treated cells based on the detergent insoluble membrane purification method. Data are presented as the ratio of p190RhoGAP in the insoluble TrX fraction to the total protein level (^*P<0.01). (C) Localization of p190RhoGAP in spread, suspended, and cytoD-, lat B-, or methyl β-cyclodextrin treated cells based on the detergent-free sucrose gradient floating assay. Data are presented as the ratio of p190RhoGAP in the lipid raft fractions (fractions 4 and 5) to total p190RhoGAP protein distributed throughout the entire gradient (^*P<0.01; ^**P<0.02).