BCR triggering leads to the PKC-dependent phosphorylation of IBtkγ. (A) DeFew cells were pretreated for 20 minutes with the PKC inhibitor Gö6976 or control DMSO vehicle at low (L, 40nM) or high (H, 200nM) concentrations; then, cells were stimulated for 10 minutes with F(ab′)2 fragments of antihuman IgM (13 μg/mL), and lysates (3 × 106 cells) were incubated for 10 minutes with FLAG-IBtkγ linked to anti-FLAG agarose beads or control anti-FLAG–agarose beads. Proteins were analyzed by Western blotting with the indicated antibodies. As control of BCR triggering, Syk levels and Tyr352-phosphorylation of Syk were analyzed with specific antibodies. One representative of 3 independent experiments with similar results is shown. (B) Primary mouse splenocytes were pretreated for 10 minutes with either the indicated PKC inhibitors (200nM), or control DMSO vehicle, followed by a 10-minute stimulation with anti–mouse IgM F(ab′)2 (20 μg/mL). Lysates (8 × 106 splenocytes) were incubated with FLAG-IBtkγ linked to anti-FLAG agarose beads or control anti-FLAG-agarose beads; proteins were analyzed by Western blotting with the indicated antibodies. One representative of 3 independent experiments with similar results is shown. (C) DeFew cells stably expressing FLAG-IBtkγ were stimulated with F(ab′)2 fragments of anti–human IgM (13 μg/mL) or left untreated in the presence or absence of Gö6976 (80nM) for the indicated time; cells were lysed, immunoprecipitated with anti-FLAG, and analyzed by Western blotting with anti-phPKCsub and anti-FLAG antibodies (top). For in vitro Btk kinase activity, protein extracts were immunoprecipitated with anti-Btk and analyzed by an ELISA-based tyrosine kinase assay. One representative of 2 independent experiments with similar results is shown. (D) The optical density (OD) of phospho-FLAG-IBtkγ was normalized according to FLAG-IBtkγ (upper bars) or Btk coimmunoprecipitated (co-IP) with FLAG-IBtkγ (lower bars).
