FIGURE 7:

CIT-K overexpression affects RhoA activation state. (A) HeLa cells were transfected with control or with CIT-K siRNA and synchronized with the thymidine/nocodazole method. Three hours after nocodazole release, active RhoA was measured by G-LISA assay (Cytoskeleton). RhoA activation was expressed as percent activation relative to control siRNA (set to 100%). (B) HeLa cells were transfected with Myc, CIT- K-Myc, or the indicated Myc-CIT-K fusion constructs and were processed as in (A). (C) HeLa cells were transfected with control or CIT-K siRNA and synchronized with the thymidine/nocodazole method. Cells were lysed 3 h after nocodazole release. Cell extracts were incubated with [³H]GDP-loaded RhoA in the presence of free GTP. After 15 min of incubation, aliquots were taken in triplicate, and the amount of bound [³H]GDP was measured. This parameter is inversely related to the RhoA-GEF activity. (D) Cell extracts prepared as in (C) were incubated with [³H]GTP-loaded RhoA in the presence of free GTP. After 15 min of incubation, aliquots were taken in triplicate, and the amount of bound [³H]GTP was measured. This parameter is inversely related to the RhoA-GAP activity. In both (C) and (D), the level of nucleotide-binding in the presence of lysis buffer was set to 100%. In every case, error bars represent SEM. Asterisks indicate that the difference is statistically significant using Student’s t test (*p < 0.05; **p < 0.01; ***p < 0.001).