Regulatory roles of RhoA and ROCK. (A) PMNs were exposed to the indicated time course of LPS. Lysates were then precleared with 15 μg GST-Sepharose (1 hour) and incubated (2 hours) with 15 μg indicated Sepharose conjugates of GST fusion protein. Precipitates were then eluted, electrophoresed on a 10% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with anti-p65. Nitrocellulose membranes were stained with Ponceau S to confirm protein loading. Blots are representative of 3 independent experiments. (B-C) PMNs were either left untreated or pretreated with Y27632 (10 μM, 60 minutes) and then exposed to buffer or to LPS (2 hours). Supernatants were tested by ELISA for TNFα protein (B) and lysates by RT-PCR for TNFα and GAPDH (C)(*P < .05 compared with LPS alone). The gel shown is representative of 3 separate experiments. (B) Error bars represent SE.