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. 2013 Feb 26;288(14):9801–9814. doi: 10.1074/jbc.M112.449231

FIGURE 1.

FIGURE 1.

Activation events in mast cells caused by 2H9 mAb. BMMCs derived from WT C57BL.6 mice were sensitized overnight with TNP-specific IgE. A, the cells were exposed to BSSA (nonactivated control, C) or activated with 2H9 mAb (10 μg/ml), SCF (100 ng/ml), or Ag (500 ng/ml TNP-BSA) for 30 min. β-Glucuronidase released into supernatant was determined as described under “Experimental Procedures.” Mean ± S.D. were calculated from 3 independent experiments performed in triplicates. B, IgE-sensitized BMMCs were loaded with Fura-2AM and exposed (arrow) to BSSA (Co.), 2H9 mAb (10 μg/ml), SCF (100 ng/ml), or Ag (500 ng/ml of TNP-BSA). Changes in [Ca2+]i were determined by spectrofluorometry as the ratio of emissions at 510 nm when the cells were excited at 340 and 380 nm. C, D, and F, IgE-sensitized BMMCs were exposed to BSSA (Co.) or activated for 3 min with 2H9 mAb (1 μg/ml), SCF (100 ng/ml), or Ag (100 ng/ml TNP-BSA). Whole cell lysates were fractionated by SDS-PAGE and analyzed by immunoblotting with phosphotyrosine-specific mAb PY-20-HRP conjugate (C), antibodies specific for the indicated phosphotyrosines, pAkt-T308 (pAktT), pAkt-S473 (pAktS), pErk-Y204 (pErkY), and pp38-Y182/T180 (pp38Y/T) (D), or antibodies specific for pSyk-Y525/526 (pSykY), or pc-Kit-Y568/570 (pc-KitY) (F). In D and F, anti-Lyn mAb (Lyn) was used as a loading control. E and G, IgE-sensitized BMMCs derived from C57BL.6 mice (E) or Lyn+/+ or Lyn−/− (G) were nonactivated (Co.) or activated with 2H9 mAb, SCF (E only), or Ag as above. The cells were solubilized in lysis buffer containing 1% Nonidet P-40 and 1% n-dodecyl-β-d-maltoside and postnuclear supernatants were immunoprecipitated (IP) with NTAL- or LAT-specific rabbit antibodies immobilized on protein A. The immunoprecipitates were analyzed by immunoblotting (IB) with phosphotyrosine-specific PY-20-HRP conjugate (PY20). Protein loading was determined by LAT- or NTAL-specific mAbs. In D-G, fold-increase in protein phosphorylation normalized to phosphorylation in nonactivated cells and protein loading is also shown. Typical results from at least 4 experiments performed are shown.