Figure 7.

Norbin-deficient neutrophils have increased fMLP-stimulated Rac and Erk activities. (A) fMLP dose response of Rac1 and Rac2 activation. Ncdn^fl/fl and Ncdn^Δmye neutrophils were stimulated with the indicated concentrations of fMLP for 10 seconds, lysed, and active, GTP-loaded Rac1 and Rac2 were isolated by PAK-CRIB pull down. Representative blots shown are from 1 of 3 independent experiments. (B) Quantification of Rac1 and Rac2 activities. Neutrophils of the indicated genotypes were stimulated with 10 μM fMLP for 10 seconds (filled symbols), or were mock-stimulated (open symbols), before PAK-CRIB pull down. Top panel: Representative western blots for Rac1 activity are shown compared with 2% of total cell lysate. Bottom panels: Rac1 and Rac2 activities were quantified by densitometry and normalized to fMLP-stimulated Ncdn^fl/fl samples. Data are mean ± standard error of the mean of 3 to 7 independent experiments; each dot is the mean of 1 experiment. Statistical analyses were conducted on log-transformed raw data by two-way analysis of variance with Dunnett’s multiple comparisons test. (C) Vav activity. Purified Ncdn^fl/fl and Ncdn^Δmye neutrophils were primed with 20 ng/mL TNF-α, 50 ng/mL GM-CSF for 45 minutes at 37°C, stimulated with 1 µM fLMP for 10 seconds (filled symbols), or mock-stimulated (open symbols), and total cell lysates underwent western blotting for active Vav and total Vav. Left-hand panel: Representative blots. Right-hand panel: quantification by ImageJ analysis of 6 independent experiments; each dot represents 1 experiment. Statistics comprised two-way analysis of variance with Šidák’s multiple comparisons test. (D) Erk activity. Purified Ncdn^fl/fl and Ncdn^‒/‒ neutrophils were primed with 20 ng/mL TNF-α, 50 ng/mL GM-CSF for 45 minutes at 37°C, stimulated with the indicated doses of fMLP for 45 seconds, and then subjected to western blotting for active and total Erk1/2. Blots shown are from 1 of 6 independent experiments. Quantification of these and related data are shown in supplemental Figure 10. Effects of the Rac inhibitor EHT 1864 (E) and Erk1/2 inhibitor SCH772984 (F) on ROS production. Neutrophils were incubated with the indicated doses of inhibitor for 1 hour during priming with TNF-α, GM-CSF, before stimulation of ROS production with fMLP as in supplemental Figure 6A. Data are mean AUC ± standard error of the mean from 4 independent experiments. Statistics comprised two-way analysis of variance with Šidák’s multiple comparisons test. Values for 50% inhibitory concentration (IC₅₀) were calculated from nonlinear best-fit curves on data normalized to maximal ROS produced.