Aggregation of FcεRI induces tyrosine phosphorylation of 3BP2 in RBL-2H3 cells.

(A) Generation of cell lines expressing HA-tagged 3BP2 wild type. The RBL-2H3 cells were stably transfected with either empty pMT3 vector or pMT3-HA-3BP2, together with pSV2-neo, by electroporation (950 μF, 310 V). Clones resistant to G418 were selected and screened by level of protein expression. Two positive cloned lines with the highest expression were chosen for further analysis. Total cell lysates of the parental RBL-2H3 and transfected cells were analyzed by immunoblotting (IB) with anti-3BP2, anti-HA, and anti-FcεRIβ antibodies used as an internal control. (B) Analysis of tyrosine phosphorylation of 3BP2. Cell lines expressing HA-3BP2 and vector-transfected control cells (pMT3) were cultured overnight with anti-DNP IgE and then stimulated with 10 ng/mL of antigen (Ag) DNP-BSA for the indicated times or incubated without antigen for 1 minute. Tyrosine phosphorylation of 3BP2 was analyzed by immunoprecipitation (IP). Cells were lysed in 1% Triton lysis buffer and cell lysates were immunoprecipitated with anti-HA antibody. Immunoprecipitates were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-pTyr mAb. The membrane was stripped and reprobed with anti-HA antibody. Similar results were obtained when the other lines were examined. (C) Tyrosine phosphorylation of endogenous 3BP2. RBL-2H3 cells sensitized with anti-DNP IgE were either unstimulated or stimulated with antigen for 1 minute. Cells were lysed in the denature buffer and cell lysates were immunoprecipitated with anti-pTyr mAb or control IgG. Immunoprecipitates were analyzed by immunoblotting with anti-3BP2 antibody. Molecular size markers are indicated at the left in kilodaltons.