RhoA negatively regulates TNFα induction in the PMN. (A) PMNs were left untreated or treated with 20 μg/mL C3 transferase (37°C, 4 hours) and then exposed either to media or to LPS (2 hours). Supernatants were tested by ELISA for TNFα protein. (*P < .05). (B) PMNs were untreated or treated with C3 transferase, exposed to buffer or LPS (1 hour) and then analyzed by RT-PCR. PMNs were also exposed to either poly-inosine/cytidine (pIC) or LPS (4 hours) and analyzed for IFN-β and GAPDH expression. The gels shown are representative of 3 experiments. (C) PMNs were left untreated or transduced with BSA or Rhotekin-RBD (25°C, 2 hours) using BioPORTER (BP). ROCKα was immunoprecipitated, and its kinase activity was assayed.²⁸  (D) PMNs were left untreated or transduced with BSA or Rhotekin-RBD (10 μg/mL, 25°C, 2 hours) using BP. Cells were then either left untreated or exposed to LPS (2 hours). Supernatants were analyzed by ELISA for TNFα protein (*P < .05 compared with buffer/unstimulated) and analyzed by RT-PCR. The gel shown is representative of 3 experiments. (E) PMNs were left untreated or exposed to 0.1% DMSO vehicle or 5 μg/mL cytochalasin B and analyzed by RT-PCR. The gel shown is representative of 4 experiments. (F) PMNs were either left untreated or transduced with wild-type or mutant WASP-CRIB and then exposed to C3 transferase (20 μg/mL, 4 hours). Lysates were analyzed by RT-PCR for TNFα and GAPDH. The gel shown is representative of 3 separate experiments. (G) PMNs were treated with buffer or C3 as in panel B and then with buffer or LPS (25 minutes). Cell sonicates were assayed for p65 activation (*P < .05 compared with NS/no oligo), and cytoplasmic fractions were immunoblotted for anti-IκBα. A representative immunoblot of 3 is shown. PCR gels are representative of 4 independent experiments. (A, D, G) Error bars represent SE.