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. 2008 Apr 21;154(5):1035–1046. doi: 10.1038/bjp.2008.148

Figure 3.

Figure 3

Roles of PKC and calcium on FcɛRI-mediated regulation of M2PK. (a) Effects of the PKC inhibitors or depletion of cellular PKC on FcɛRI-mediated mast cell degranulation. RBL-2H3 cells were treated with either Gö6973 or Gö6983 for 20 min, or with 1 μM PMA overnight, then stimulated with 1 μg mL−1 DNP-BSA for 10 min. ***P<0.001 compared with the vehicle-treated group (DMSO). The inhibitions of mast degranulation by PKC inhibitors or PMA treatment were statistically significant (P<0.001) compared with the vehicle-treated group at all the concentrations higher than 0.5 μM PKC inhibitors and 0.1 μM PMA. (b) Effects of the PKC inhibitors or depletion of cellular PKC on the FcɛRI-mediated inhibition of M2 type PK activity. The cells were treated with increasing concentrations of Gö6973 or Gö6983 for 20 min or with 1 μM PMA overnight, and then stimulated with 100 ng mL−1 DNP-BSA for 5 min. PK activity was measured as described under Materials and methods. ***P<0.001 compared with the control (DMSO/DNP-BSA) group. (c) Effects of calcium on mast cell degranulation. For the calcium-free experimental group (shown as X), a PIPES buffer without CaCl2 was used. Cells were stimulated with 1 μg mL−1 DNP-BSA for 10 min. ***P<0.001 compared with the calcium (O) group. (d) Effects of calcium on the enzymatic activities of M2PK. Cells were stimulated with 100 ng mL−1 DNP-BSA for 5 min. ***P<0.001 compared with the calcium (O)/control group; #P<0.1 compared with calcium (O)/DNP-BSA group. (e) Effects of calcium on the tyrosine phosphorylation of Syk and M2PK. The RBL-2H3 cells were treated with 100 ng mL−1 DNP-BSA for 1 min in the presence or absence of calcium. The cell lysates were immunoprecipitated with the PY-20 antibody conjugated to agarose beads and the immunoprecipitates were analysed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The blots were probed with the antibodies to Syk and M-type PK at 1:1000 and 3000 dilutions, respectively. Data represent results from two independent experiments with similar outcomes. BSA, bovine serum albumin; DMSO, dimethylsulphoxide; FcɛRI, high-affinity IgE receptor; M2PK, M2-type pyruvate kinase; PKC, protein kinase C; PMA, phorbol myristate acetate; RBL-2H3, rat basophilic leukaemia cells.