MT depolymerization induces GEF-H1–dependent actin polymerization and MLC phosphorylation. (A) GEF-H1^+/+ and GEF-H1^−/− neutrophils were either untreated or pretreated with10 µM Y-27632 for 30 min before stimulation with DMSO or nocodazole (Noc) for the indicated times. The geometric MFI of phalloidin stained cells was determined by flow cytometric analysis of 30,000 gated events in triplicate. Data were pooled from at least four independent experiments, and the mean ± SEM of the percentage increase in F-actin relative to DMSO-treated cells is indicated. (n = 4; *, P < 0.05; **, P < 0.005). (B) GEF-H1^+/+ and GEF-H1^−/− neutrophils were either untreated or pretreated with 10 µM Y-27632 before stimulation with 10 µM DMSO or nocodazole for the indicated times. Antibodies used for immunoblotting are indicated. Results are combined from two concurrent Western blots, indicated by a dotted line. (C) GEF-H1^+/+ and GEF-H1^−/− neutrophils were treated with 10 µM fMLP for 1 min, 10 ng/ml CXCL1 for 5 min, 1 µg/ml C5a for 2 min, or DMSO as a control. Cell lysates were immunoblotted with the indicated antibodies.