Figure 4.

(A) G2A induces transcriptional activation of SRF. NIH 3T3 cells were cotransfected with reporter plasmid pSRF-Luc, pTK-Renilla Luc, and pEXV3 G2A or pEXV3 GFP with or without pRK5 mycC3T (totaling 0.9 μg DNA). Twenty four hours after transfection, cells were lysed and subjected to luciferase assays. Transfections and SRF-Luc assays were performed in triplicate, and results presented are typical of three independent experiments. (B) G2A activates RhoA in Swiss 3T3 cells. Swiss 3T3 cells were infected with MSCV GFP or MSCV G2AiresGFP retroviruses and 24 h later were serum-starved for 12 h. Cells subsequently were lysed, and lysates were incubated on ice for 1 h with glutathione S-transferase-RRBD immobilized on glutathione-agarose beads to affinity precipitate RhoA-GTP. Affinity precipitates (RhoA GTP) and aliquots of total lysates (total RhoA) were Western blotted with a mAb against RhoA. Lanes 2 and 4, lysophosphatidic acid (LPA) (1 μg/ml, 5 min) stimulated activation of RhoA. Lanes 1 and 3, Increased levels of RhoA GTP in G2A expressing Swiss 3T3 cells compared with control Swiss 3T3 cells.