Integrin-dependent events are up-regulated in ARAP3-deficient neutrophils. (A) Integrin-dependent signaling. Bone marrow–derived neutrophils were prepared from mock (WT) and tamoxifen-induced (KO) Arap3^fl/flERT2Cre⁺ mice. Cells (5×10⁻⁶) were left to adhere to hiFCS- or polyRGD-coated tissue culture plastic to induce intracellular signaling before being scraped into lysis buffer. Lysates were subjected to SDS-PAGE and immunoblotted for phospho-PKB (Ser473), phospho-p38 (Thr180 and Tyr182), and β-COP as a loading control. A representative blot is shown on the left. Blots were quantified using ImageJ v1.37 software, and pooled data from 3 independent experiments are plotted (top right, phospho-PKB; bottom right, phospho-p38), expressed as a percentage of the response obtained with mock-induced neutrophils plated onto polyRGD (data ± SEM). (B) Gelatinase granule release. Neutrophils were prepared as in panel A, and plated in wells that had been coated with hiFCS or fibrinogen in the presence of 20 ng/mL of TNFα. For a positive control, neutrophils were stimulated with 1μM fMLF in the presence of 10μM cytochalasin B (CB). Supernatants from all wells were used for zymography. Stained gels were quantified using ImageJ v1.37 software. A representative stained zymography gel is shown on the left. The positive control samples were not loaded immediately adjacent to adhesion-induced samples. For ease of viewing, the 2 parts of the gel were pasted here, as indicated by a dotted line. Pooled data from 3 independent experiments are plotted (data ± SEM) on the right. (C) Adhesion and spreading. Neutrophils were prepared as in panel A, and 5 × 10⁶ cells were plated into 24-well plates that had been coated with 20 μg/mL of polyRGD, left to adhere for 10 minutes, and fixed. In washed plates, adherent (phase dark) and spread cells (phase light) were counted in 4 randomly chosen fields of view from each individual experiment, and the percentage of spread cells of total numbers was determined. Example photos are shown on the left; on the right are plotted data combined from 3 independent experiments (data ± SEM). Statistical analysis was by paired t tests performed on the raw data. *P < .05; **P < .01; ***P < .001. (D) Ex vivo flow chamber studies were performed with tamoxifen-induced Arap3^fl/flERT2Cre⁺ (iko) and wild-type animals (wt) using microflow chambers (0.4 × 0.04 mm) coated with rmE-selectin (20 μg/mL), rmCXCL1 (15 μg/mL), and rmICAM-1 (15 μg/mL). Flow chambers were recorded for 10 minutes, and adhesion efficiency (adherent cells/mm² × WBC) was calculated (data ± SEM; n = at least 5 chambers per group).