Figure 3.

Cotransfection of RhoAV14, but not RhoAN19, RacV12, or CDC42V12, with radixin is sufficient to cause relocalization to apical membrane/actin protrusions in NIH3T3 cells. NIH3T3 cells were transiently transfected with radixin (a and b), radixin and RhoAV14 (c and d), radixin and RhoAN19 (e and f), radixin and RacV12 (g and h), and radixin and CDC42V12 (b). All GTPase constructs were myc epitope tagged. HA-radixin (a, b, c, e, and g) and myc-tagged GTPases (d, f, and h) localizations are shown. Cotransfections followed by immunolocalization were performed in 12 independent experiments. Radixin localization in the presence of activated RhoA was further verified by transfection of radixin into two independent NIH3T3 cell lines stably expressing the RhoAV14 allele. In all experiments, at least 100 transfected cells were examined for each condition, and cells shown are representative of > 80% of the transfected cell population. In the presence of activated RhoA, but not dominant negative RhoA, activated Rac, or activated CDC42, radixin localized into apical membrane protrusions. α-myc and actin staining were used in all experiments to verify functional expression of RhoAV14. Bar, 10 μm.