Phosphorylation of PI3Kγ requires Ca²⁺ and is PKCβ-dependent.

<p>(A) Stimulus-induced phosphorylation of endogenous p110γ on Ser582 in wild type BMMCs. IL-3 deprived cells were stimulated with 100 nM PMA, 1 µM thapsigargin, 1 µM adenosine, or 20 ng/ml DNP for 2 min. Where indicated (IgE), BMMCs were loaded with IgE (100 ng/ml) overnight. PI3Kγ was immunoprecipitated from cell lysates with an anti-PI3Kγ antibody, before precipitated protein was probed for phosphorylated p110γ (pp110γ) with a phospho-specific anti-pSer582 antibody (validation of antibody see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio.1001587.s004" target="_blank">Figure S4</a>). PI3Kγ phosphorylation is shown normalized to total PI3Kγ levels (mean ± standard error of the mean [SEM], <i>n</i> = 3; * depict analysis using unstimulated control. & reference point is IgE only). (B) IgE/antigen-induced Ser582 phosphorylation of p110γ requires Ca²⁺ influx. Cells were stimulated as in (A), but exposed to EDTA, EGTA, or loaded with BAPTA/AM where indicated (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001587#pbio-1001587-g002" target="_blank">Figure 2</a>). Phosphorylated p110γ was detected as in (A); mean ± SEM, <i>n</i> = 3; * comparison with unstimulated control; ^&analysis of stimulated versus chelator treated). (C) Phosphorylation of p110γ in wild type and PKCβ^−/− BMMCs. Experimental settings were as in (A), and (D) depicts quantification of pp110γ in relation to total p110γ protein (mean ± SEM; PMA <i>n</i> = 4, antigen <i>n</i> = 3). Cells devoid of p110γ were included as negative control.