by Katsushi Miura, and Donald W. MacGlashan Phosphatidylinositol-3 kinase regulates p21ras activation during IgE-mediated stimulation of human basophils by Katsushi Miura, and Donald W. MacGlashan Blood Volume 96(6):2199-2205 September 15, 2000 ©2000 by American Society of Hematology

Effect of LY294002 on release of histamine and LTC4 and phosphorylation of MEK1 and ERKs after stimulation with anti-IgE Ab or FMLP.(A) Effect of LY294002 on release of histamine and LTC4. Effect of LY294002 on release of histamine and LTC4 and phosphorylation of MEK1 and ERKs after stimulation with anti-IgE Ab or FMLP.(A) Effect of LY294002 on release of histamine and LTC4. Basophils were preincubated with DMSO (1:10 000 dilution) or LY294002 (10 μmol/L) for 10 minutes and were stimulated with or without FMLP (1 μmol/L) for 5 minutes or anti-IgE Ab (0.5 μg/mL) for 10 minutes. Reactions were stopped with the addition of ice-cold PAG, and the cells were microfuged. Supernatants were collected for histamine (closed column) and LTC4 (open column) measurements (n = 3). (B) Effect of LY294002 on phosphorylation of MEK1 and ERKs. Cell pellets were lysed and subjected to Western blot analysis as described in “Materials and methods.” The anti-MEK1 blot indicated essentially equal protein loading (data not shown). The Western blot shown is representative of 3 separate experiments. Katsushi Miura, and Donald W. MacGlashan, Jr Blood 2000;96:2199-2205 ©2000 by American Society of Hematology

Effect of LY294002 on activation of ras after stimulation with anti-IgE Ab.Basophils were resuspended in PAGCM (containing 1 mmol/L Ca++) or PAG (without Ca++) and preincubated with or without LY294002 (10 μmol/L) for 10 minutes. Effect of LY294002 on activation of ras after stimulation with anti-IgE Ab.Basophils were resuspended in PAGCM (containing 1 mmol/L Ca++) or PAG (without Ca++) and preincubated with or without LY294002 (10 μmol/L) for 10 minutes. The cells were stimulated with or without anti-IgE (0.5 μg/mL) for 5 minutes. Reactions were stopped with the addition of ice-cold PAG, and the cells were microfuged. Clarified lysates were subjected to affinity precipitation with RBD-GST. Affinity-precipitated GTP-ras was detected by immunoblotting with anti-ras Ab. The immunoblot shown is representative of 2 separate experiments. Katsushi Miura, and Donald W. MacGlashan, Jr Blood 2000;96:2199-2205 ©2000 by American Society of Hematology

Effect of LY294002 on the [Ca++]i response after stimulation with anti-IgE Ab.Basophils were labeled with fura-2/am. Effect of LY294002 on the [Ca++]i response after stimulation with anti-IgE Ab.Basophils were labeled with fura-2/am. After washing, the cells were treated with or without LY294002 for 10 minutes and stimulated with anti-IgE Ab (0.5 μg/mL). The [Ca++]i response was analyzed as described in “Materials and methods.” The result shown is the average of 2 separate experiments. Katsushi Miura, and Donald W. MacGlashan, Jr Blood 2000;96:2199-2205 ©2000 by American Society of Hematology

Effect of LY294002 on phosphorylation of syk and shc and association of Grb2 and shc after stimulation with anti-IgE Ab.Basophils were preincubated with DMSO (1:10 000 dilution) or LY294002 (10 μmol/L) for 10 minutes and were stimulated with anti-IgE (0.5 μ... Effect of LY294002 on phosphorylation of syk and shc and association of Grb2 and shc after stimulation with anti-IgE Ab.Basophils were preincubated with DMSO (1:10 000 dilution) or LY294002 (10 μmol/L) for 10 minutes and were stimulated with anti-IgE (0.5 μg/mL) for 5 minutes. Reactions were stopped with the addition of ice-cold PAG, and the cells were microfuged. Clarified lysates were immunoprecipitated with anti-syk (A), anti-shc (B), or anti-Grb2 (C). The immunoprecipitated proteins were subjected to Western blot analysis with the indicated Abs, as described in “Materials and methods.” The same membranes were stripped and reblotted with the indicated Ab. The anti-syk (A), anti-shc (B), and anti-Grb2 (C) blots indicate essentially equal protein loading. Each Western blot shown is representative of 2 separate experiments. Katsushi Miura, and Donald W. MacGlashan, Jr Blood 2000;96:2199-2205 ©2000 by American Society of Hematology

Tyrosine phosphorylation of p85 after stimulation with anti-IgE Ab and FMLP, and the effect of LY294002 or PP1.Basophils were stimulated with or without anti-IgE (0.5 μg/mL) (A) or FMLP (1 μmol/L) (B) for the times indicated. Tyrosine phosphorylation of p85 after stimulation with anti-IgE Ab and FMLP, and the effect of LY294002 or PP1.Basophils were stimulated with or without anti-IgE (0.5 μg/mL) (A) or FMLP (1 μmol/L) (B) for the times indicated. (C) Basophils were pretreated with or without DMSO, LY294002 (10 μmol/L), or PP1 (10 μmol/L) for 10 minutes and stimulated by anti-IgE Ab for 5 minutes. Reactions were stopped with the addition of ice-cold PAG, and the cells were microfuged. Clarified lysates were immunoprecipitated with anti-p85 Ab. The immunoprecipitated proteins were subjected to Western blot analysis with antiphosphotyrosine Ab as described in “Materials and methods.” The same membranes were stripped and reblotted with anti-p85 Ab. The anti-p85 blot indicates essentially equal protein loading. Each Western blot shown is representative of 2 separate experiments. Katsushi Miura, and Donald W. MacGlashan, Jr Blood 2000;96:2199-2205 ©2000 by American Society of Hematology

Effect of Ro-31-8220 on phosphorylation of ERKs after stimulation with anti-IgE Ab or FMLP.Basophils were preincubated with DMSO (1:10 000 dilution) or Ro-31-8220 (1 μmol/L) for 10 minutes and were stimulated with or without anti-IgE Ab (0.5 μg/mL) for 10 m... Effect of Ro-31-8220 on phosphorylation of ERKs after stimulation with anti-IgE Ab or FMLP.Basophils were preincubated with DMSO (1:10 000 dilution) or Ro-31-8220 (1 μmol/L) for 10 minutes and were stimulated with or without anti-IgE Ab (0.5 μg/mL) for 10 minutes or PMA (50 ng/mL) for 20 minutes. Reactions were stopped with the addition of ice-cold PAG, and the cells were microfuged. Cell pellets were lysed and subjected to Western blot analysis as described in “Materials and methods.” The anti-ERK1 and anti-ERK2 blots indicated essentially equal protein loading (data not shown). The Western blot shown is representative of 2 separate experiments. Katsushi Miura, and Donald W. MacGlashan, Jr Blood 2000;96:2199-2205 ©2000 by American Society of Hematology