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. 2005 Oct 10;102(42):15207–15212. doi: 10.1073/pnas.0504501102

Fig. 2.

Fig. 2.

In vivo RhoA activity in transiently transfected HEK293. (A) RhoA pull-down assay. No detectable RhoA activity was observed in DLC2α- and DLC2β-transfected cells. Significant reduction in RhoA activity was observed in DLC2γ-transfected cells as compared with the mock-transfected and parental HEK293 cells. In the DLC2γ GAP mutant-transfected cells DLC2γ K618E and DLC2γ R622E, the RhoA activity was similar to that of the mock-transfected and parental cells. The RhoA activity of a RhoDA (dominant active)-transfected cell was taken as a positive control of the RhoA activity assay. The protein expression of DLC2 was confirmed by anti-GFP Ab, and the RhoA expression in the total cell lysate was probed as a loading control. GST protein was used as a negative control. (B) The in vivo RhoA activity decreased with increased amounts of DLC2γ. (C) Effects of actin cytoskeleton in Swiss 3T3 transfected with DLC2γ, DLC2γ K618E, and DLC2γ R622E. There was rounding off of the DLC2γ-transfected cells with reduced stress fiber formation. In contrast, no change in cell morphology or actin stress formation was seen in cells transfected with the DLC2 GAP-mutants.