KC-triggered rapid activation of VLA-4 adhesiveness does not involve β-arr–dependent early activation of PI3K and ERK1/2 MAPKs. (A-C) Effect of either β-arr isoform depletion on KC-induced activation of Akt and ERK1/2. (A) Cells were stimulated with 0.5 μg/mL KC for the indicated times. Phosphorylated Akt (p-Akt) and phosphorylated ERK1/2 (p-ERK1/2) were detected by immunoblotting with phospho-specific antibodies. The levels of total Akt, ERK1/2, and β-actin in whole-cell lysates were used as protein loading control. (B-C) Quantitative analysis of KC-stimulated activation of Akt (B) and ERK1/2 (C) in control and β-arr–depleted CXCR2⁺ RBL-2H3 cells. The levels of p-Akt and p-ERK1/2 were quantified by densitometry and normalized to the total Akt and ERK1/2, respectively. Values at each time point represent the percentage of normalized p-Akt (B) and p-Erk1/2 (C) compared with siRNA control-transfected cells. Data are expressed as mean ± SEM of 3 independent experiments (*P < .05, **P < .01, ***P < .001 relative to control RBL-2H3 cells at the same time point). (D) Effect of inhibitors of PI3K and MAPKs on frequency and strength of tethers mediated by CXCR2⁺ RBL-2H3 cells with a low concentration (0.025 μg/mL) of VCAM-1/Fc coimmobilized with heat-inactivated (data not shown) or functional KC (2 μg/mL) under flow conditions. Data are expressed as mean plus or minus the range of 2 experimental fields. Results are representative of 3 independent experiments. (E-H) Effect of inhibitors of PI3K and MAPKs on KC-induced activation of Akt, ERK1/2, and Rap1. (E) CXCR2⁺ RBL-2H3 cells were pretreated for 30 minutes at 37°C with either vehicle control, 15 μM LY294002, or 10 μM U0126 before addition of 0.5 μg/mL KC for the indicated times. p-Akt and p-ERK1/2 were detected as described above. Rap1-GTP was determined in pull down assays as described in the legend to Figure 4A. (F-H) Quantitative analysis of KC-stimulated activation of Akt (F), ERK1/2 (G), and Rap1 (H) in vehicle- and inhibitor-treated CXCR2⁺ RBL-2H3 cells. The levels of p-Akt and p-ERK1/2 were quantified as described above. The levels of Rap1-GTP were quantified as described in the legend to Figure 4B. Values at each time point represent the percentage of normalized p-Akt (F), p-ERK1/2 (G), and Rap1-GTP (H) relative to vehicle-treated cells. Data are expressed as mean ± SEM of 3 independent experiments (*P < .05, **P < .01 relative to vehicle-treated RBL-2H3 cells at the same time point).