GEF-H1 relocalizes to uropods immediately after exposure to shear stress. (A) HL-60 cells were exposed to static or shear stress conditions and were immunolabeled with antitubulin, anti–GEF-H1, or anti–pS885-specific antibodies as indicated. Red boxes highlight MT and non–MT-associated pools of GEF-H1. Bar, 5 µm. (B) A detail from (A) highlighting MT and non–MT-associated pools of GEF-H1. Red bars indicate where the MFIs of pS885 and GEF-H1 were measured. The mean ratio of pS885/GEF-H1 ± SEM was determined from 6–10 random polarized cells in each of five separate experiments. Bar, 1 µm. (C) HL-60 cells were plated on ICAM-coated surfaces and exposed to 2 dynes/cm² of shear stress for 20 s or left in shear-free conditions, fixed and stained with the indicated antibodies. Arrow indicates direction of shear. Bar, 5 µm. (D) Localization of GEF-H1 in uropods was determined based on colocalization with flotillin-2. 30 random spread cells were scored per experiment. The means ± SEM of the percentage of cells with uropod-associated GEF-H1 were determined from at least five independent experiments. (E) GEF-H1^+/+ and GEF-H1^−/− neutrophils were imaged at 30 min after stimulation with 10 µM nocodazole by phase-contrast microscopy at 63× magnification. Arrowheads indicate uropods. Bar, 5 µm. (F) Neutrophils were untreated or pretreated for 30 min with 10 µM Y-27632 (Y276), 100 µM blebbistatin (Bleb), or 10 µM latrunculin A (LatA), followed by an additional 30-min incubation with DMSO or 10 µM nocodazole. At least 100 cells were scored per experiment. Data are presented as mean ± SEM and are representative of three independent experiments. (G) Neutrophils were treated with DMSO or nocodazole (Noc) for 30 min and then fixed and labeled for F-actin (red) and pMLC (green). Cells were imaged by confocal fluorescence microscopy at 60× magnification. Bar, 5 µm.