Cdc42 controls neutrophil polarity via WASp. (A) WT and Cdc42^−/− neutrophils were un-stimulated or stimulated with fMLP and plated on Fg-coated slides for 10 minutes. Western blots from whole-cell lysate (WCL), probed with p-WASp (Y-291) and total WASp. Representative blot of 3 independent experiments (mean ± SD; **P = .0059). (B) Immunofluorescence of WASp and F-actin in fMLP and Fg-stimulated neutrophils. Arrow indicates the tail of neutrophils identified on phase contrast images based on the classic morphology of the tail at the uropod (in all the subsequent images). Histogram is percent of cells exhibiting WASp enriched at uropod (mean ± SD; *P = .0097; 3 independent experiments). (C) WTCdc42 and Cdc42S71P proteins were subjected to the pull down assay to assess ability of the protein to bind WASp or PAK. The amount of immunoprecipitated GTP-Cdc42 is revealed by immunoblot with anti-HA. WCL blotting was used for equal input (lowest blot). (D) Immunoblot of WCL for expression of wtCdc42 and Cdc42S71P in Cdc42^−/− neutrophils and for phosphorylation of PAK (p-PAK1) and WASp (pWASp). P-PAK and pWASp blots were performed independently and actin loading control is shown for each. (E) Neutrophil migration was examined by time lapse video microscopy in gradient of fMLP and on surface coated with fibrinogen, in Zigmond chamber. Measurement of straightness of migration was performed in ImageJ Version 1.43J software. Scatter plot of individual cells (*P < .05, **P < .01; 3 independent experiments). (F) Immunofluorescence analysis of F-actin distribution on stimulation with fMLP and Fg, F-actin was labeled with Rhodamine Phalloidin and slides were mounted in SlowFade Gold Antifade. (G) Histogram is percent of cells exhibiting multiple F-actin protrusions as seen in panel F (mean ± SD; **P < .001; 3 independent experiments). Fluorescence images were captured at room temperature using a Leica DMI6000 fluorescence microscope at 63× objective N/A1.3 with ORCA-ER C4742-95 camera driven by Openlab Version 5.5.0 software.