![Figure 3. Overexpression of GEM-Cbl-b suppresses FcϵRI-mediated Ca++ mobilization and degranulation in RBL-2H3 cells. (A) Analysis of FcϵRI-mediated β-hexosaminidase release. Control cells, GEM-Cbl-b or GEM-Cbl-b (C373A) overexpressing cells were cultured overnight with anti-DNP IgE and then stimulated with the indicated concentrations of antigen DNP-BSA (ng/mL) or Ca++ ionophore A23187. The antigen-induced β-hexosaminidase release was presented as a percentage of the A23187-induced maximal β-hexosaminidase release. The A23187-induced average releases ± SD as a percentage of total activity were 42% ± 2% (ctrl.), 37% ± 1% (GEM-Cbl-b), and 34% ± 1% (GEM-Cbl-b [C373A]), respectively. The results are the mean values ± SD from 3 independent experiments. The results were representative of 10 experiments. (B) FcϵRI-mediated Ca++ mobilization is suppressed by the overexpression of GEM-Cbl-b. Intracellular free Ca++ concentration ([Ca++]i) was measured by means of fluorescent indicator fura-2. IgE-sensitized control cells and cells overexpressing GEM-Cbl-b or GEM-Cbl-b (C373A) were loaded with fura-2 and then activated with either 30 ng/mL antigen (top row) or 1 μM thapsigargin (bottom row). The traces show the 340:380 fluorescence ratios. (C-D) Overexpression of GEM-Cbl-b suppresses tyrosine phosphorylation of PLC-γ. Control cells and cells overexpressing GEM-Cbl-b or GEM-Cbl-b (C373A) were primed with anti-DNP IgE and stimulated with 30 ng/mL antigen DNP-BSA for the indicated times. Cell lysates (107) were solubilized in 1% Triton lysis buffer and immunoprecipitated with anti–PLC-γ1 mAb (C) or anti–PLC-γ2 antibody (D). The immunoprecipitates were separated by SDS-PAGE and analyzed by the immunoblotting with anti-pTyr mAb along with anti–PLC-γ1 mAb (C) or anti–PLC-γ2 antibody (D). The numbers at the bottom of the figures are the normalized densitometric analysis of PLC-γ1 and PLC-γ2 tyrosine phosphorylation. Similar results were obtained when the other cloned lines were examined. The results were representative of 3 experiments (B-D).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/103/5/10.1182_blood-2003-07-2260/6/zh80050457740003.jpeg?Expires=1735831341&Signature=HRxWuynWCWG09cmnT35IlPfBxTv5JKCwQL-Al2ak23sFYRrUXdk~LDAiZfVAq7iR5UL8LKhAmb~DK0rY6QkKG4uA6fzYPZJbFnFHRbgfO3QGvNWevvSf80rnApVWH-p7HekpyBLfHI-5GiQcoWqECEJS4n4t7ECFzPd7qNn-ME0kDCArP2bSs7qXjCv3~L7FruLNdIhUQWRYA-Zh4EXb1EJfVhWIie3ZYxMDAu9k9tt7z7dMAo~C5I5XndbQY2s5yVLymAmVGj1iQKHGL5B2BZggFxHlhJ9v-yfAW1awX4f~3vs9q7IWK1uVrgcUFVyu6wJGZdkuIUgUc7Q9OdhwcQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Overexpression of GEM-Cbl-b suppresses FcϵRI-mediated Ca++ mobilization and degranulation in RBL-2H3 cells. (A) Analysis of FcϵRI-mediated β-hexosaminidase release. Control cells, GEM-Cbl-b or GEM-Cbl-b (C373A) overexpressing cells were cultured overnight with anti-DNP IgE and then stimulated with the indicated concentrations of antigen DNP-BSA (ng/mL) or Ca++ ionophore A23187. The antigen-induced β-hexosaminidase release was presented as a percentage of the A23187-induced maximal β-hexosaminidase release. The A23187-induced average releases ± SD as a percentage of total activity were 42% ± 2% (ctrl.), 37% ± 1% (GEM-Cbl-b), and 34% ± 1% (GEM-Cbl-b [C373A]), respectively. The results are the mean values ± SD from 3 independent experiments. The results were representative of 10 experiments. (B) FcϵRI-mediated Ca++ mobilization is suppressed by the overexpression of GEM-Cbl-b. Intracellular free Ca++ concentration ([Ca++]i) was measured by means of fluorescent indicator fura-2. IgE-sensitized control cells and cells overexpressing GEM-Cbl-b or GEM-Cbl-b (C373A) were loaded with fura-2 and then activated with either 30 ng/mL antigen (top row) or 1 μM thapsigargin (bottom row). The traces show the 340:380 fluorescence ratios. (C-D) Overexpression of GEM-Cbl-b suppresses tyrosine phosphorylation of PLC-γ. Control cells and cells overexpressing GEM-Cbl-b or GEM-Cbl-b (C373A) were primed with anti-DNP IgE and stimulated with 30 ng/mL antigen DNP-BSA for the indicated times. Cell lysates (107) were solubilized in 1% Triton lysis buffer and immunoprecipitated with anti–PLC-γ1 mAb (C) or anti–PLC-γ2 antibody (D). The immunoprecipitates were separated by SDS-PAGE and analyzed by the immunoblotting with anti-pTyr mAb along with anti–PLC-γ1 mAb (C) or anti–PLC-γ2 antibody (D). The numbers at the bottom of the figures are the normalized densitometric analysis of PLC-γ1 and PLC-γ2 tyrosine phosphorylation. Similar results were obtained when the other cloned lines were examined. The results were representative of 3 experiments (B-D).
