Overexpression of 3BP2-SH2 domain suppresses FcεRI-mediated degranulation in RBL-2H3 cells.

(A) Generation of cell lines expressing the HA-tagged SH2 domain of 3BP2. The RBL-2H3 cells were stably transfected with pMT3-HA-3BP2-SH2 (454-559) by electroporation. G418-resistant clones were screened by immunoblotting and 2 positive cloned lines with highest expression were selected for further analysis. Total cell lysates of the transfected cell lines were analyzed with anti-HA and anti-FcεRIβ antibodies used as an internal control. (B) Analysis of FcεRI-mediated β-hexosaminidase release. Control cells transfected with pMT3 vector alone and cells overexpressing 3BP2-SH2 domain were cultured overnight with anti-DNP IgE and then stimulated with the indicated concentrations of the antigen DNP-BSA (ng/mL) or with the calcium ionophore A23187 (μM). The antigen- or A23187-induced releases are normalized by expression as a percentage of the total β-hexosaminidase activity. (C) Analysis of thapsigargin-induced β-hexosaminidase release. Cells were stimulated with the indicated concentrations of thapsigargin (μM); releases are normalized by expression as a percentage of β-hexosaminidase activity induced by 1 μM A23187. The results are the mean values ± SE from 3 independent experiments.