Functional impact of RHOA mutants. (A-D) Kinetics of GDP/GTP-binding for different RHOA mutants in the presence of 0.8 mM Mg²⁺. (A-B) 5 µM of radio-labeled GTP analog ([³⁵S]GTPγS) was incubated with indicated RHOA mutants, and the amounts of RHOA-bound [³⁵S]GTPγS at indicated time points are plotted. (C-D) RHOA-bound [³⁵S]GTPγS and [³H]GDP were measured at indicated time points after their dissociation was initiated by the addition of unlabeled GTPγS. Data represent mean ± standard deviation (n = 3 for each group). The significance of difference was determined by two-tailed unpaired Student t test. *P < .05; **P < .005; ***P < .0005; 3 independent experiments. (E) Activity of the SRF-RE reporter in 293T cells transduced with indicated RHOA mutants in luciferase assay. The differences between mutants and the WT RHOA were all statistically significant (*P < .05; **P < .005). Data represent mean ± standard deviation (n = 4). P value was determined using Student t test. Three independent experiments were performed.