Figure 7

Increase in the endogenous RhoA activation in NIH 3T3 cells monitored by the Rhotekin assay. Upper panel: Rhotekin pull-down of RhoA•GTP from cells ectopically expressing full-length wild type (wt) PRG, the isolated DH-PH tandem and the 3R and 4R mutants of the full-length form. The cells were under serum free conditions. Bottom panel: quantitative assessment of RhoA activation using a set of three independent experiments. The isolated DH-PH tandem is not included because the expression level was significantly higher than that of the three full-length PRG variants, and while the increased endogenous RhoA activity as measured by the Rhotekin assay (see Methods Section), demonstrating that these expressed constructs are biologically active in cells. Mutant PRG-RhoGEFS were FLAG tagged. Western blotting for FLAG indicated that the mutants were expressed at an equal level while the DH-PH domain was overexpressed (data not shown) accounting for the greater level of active RhoA.