Phosphorylation of p40^phox on T154 in response to fMLP and IgG-SRBCs is partially dependent on cPKCs, whereas phosphorylation of p40^phox in response to PMA and S aureus is mediated by PKCδ. (A) Stimulus-elicited phosphorylation of p40^phox. BMNs from wt C57Bl/6J mice (5 × 10⁵/sample) were incubated with fMLP, S aureus, IgG-SRBCs, or PMA. Assays were terminated at the indicated time points by placing samples on ice with immediate addition of an excess of ice-cold buffer. Cells were pelleted and sonicated into SDS sample buffer. Proteins were resolved by SDS-PAGE, after which immunoblotting was performed using an antibody directed against p40^phox phosphorylated on T154 or total p40^phox as a loading control. A representative blot from 2 independent experiments is shown for each condition. (B) BMNs from wt C57Bl/6J mice were pretreated with vehicle control (0.1% dimethyl sulfoxide), BIM-1, Gö 6976, or Gö 6983 at a concentration of 1μM or with wortmannin (Wort) at a concentration of 100 nM or 300 nM. Cells were stimulated with fMLP for 10 seconds, S aureus or IgG-SRBCs for 20 minutes, PMA for 10 minutes. Assays were stopped, and cells were immediately processed as described in panel A. Quantification of levels of phospho-p40^phox and total p40^phox was performed using Aida software. Histograms represent ratios of phospho-p40^phox:total p40^phox as a percentage of vehicle control. Data are means ± SEM (n = 3 independent experiments for each agonist, performed in duplicate). (C) wt or PKCδ^−/− neutrophils were stimulated with fMLP for 10 seconds, S aureus for 20 minutes, PMA for 10 minutes. Assays were stopped, and cells were processed as described in A. Quantification of levels of phospho-p40^phox and total p40^phox was performed using Aida software. Histograms represent ratios of phospho-p40^phox:total p40^phox as a percentage of wt stimulated BMNs. Data are means ± range (n = 2) for fMLP and means ± SEM (n = 3) for S aureus and PMA.