Dominant-active Cdc42 and Rac enhance secretion, whereas dominant-negative Cdc42 and Rac inhibit secretion. (a) RBL-2H3 cells were infected with vaccinia virus containing empty vector (lanes 2 and 6), or a vector expressing Cdc42 (lane 3), Cdc42V12 (lane 4), Cdc42N17 (lane 5), Rac (lane 7), RacV12 (lane 8), or RacN17 (lane 9) for 6 h at 37°C, or were uninfected (lane 1). Proteins were solubilized and resolved by SDS-PAGE. Cdc42 and Rac were detected by Western blotting using either anti-Cdc42 or anti-Rac antibody, respectively. Virus-expressed proteins migrate at a slightly higher molecular mass compared with the endogenous protein due to the presence of the myc epitope. (b) β-Hexosaminidase secretion was measured in RBL-2H3 cells infected with the different forms of Cdc42 and Rac upon stimulation with 100 ng/ml DNP/BSA for 1 h at 37°C and under nonstimulated conditions. Degranulation is given as the percent of total cellular β-hexosaminidase released, as measured by treatment of RBL-2H3 cells in parallel with 0.5% Triton X-100. Error bars represent the standard deviation of four independent experiments. The paired t test was used to evaluate the statistical significance between cells infected with empty vector and Cdc42 (0.0264), empty vector and Cdc42N17 (0.0023), empty vector and RacN17 (0.0191), Cdc42 and Cdc42N17 (0.0437), and Rac and RacN17 (0.0424). A score of ≤0.05 denotes statistical significance. The comparison between cells infected with empty vector and Rac was not statistically significant.