CIN85 associates with Cbl and BLNK and regulates their phosphorylation in B cells. (A) BJAB cells stably expressing either WT or SH3-deleted CIN85 were stimulated with 20 μg/mL of F(ab′) 2 goat anti–human IgM for the indicated time periods. Immunoprecipitates with anti-V5 or anti-BLNK mAb were separated on a 10% SDS-PAGE gel and analyzed by Western blotting with anti–c-Cbl, anti–Cbl-b, anti-BLNK, or anti-V5 mAb. 5′c, immunoprecipitation of the cell lysates at 5 minutes with isotype control. (B) Control BJAB cells and stable transformants expressing either WT or SH3-deleted CIN85 were stimulated with 20 μg/mL of F(ab′) 2 goat anti–human IgM for the indicated time periods. Immunoprecipitates with anti–c-Cbl mAb were separated on a 10% SDS-PAGE gel and analyzed by Western blotting with anti-phosphotyrosine or anti–c-Cbl mAb. The resulting values are expressed as fold changes in protein expression compared with unstimulated control cells. The values are the mean ± SD of 3 independent experiments (*P < .05, **P < .01 vs controls). (C) Control BJAB cells and stable transformants expressing either WT or SH3-deleted CIN85 were stimulated with 20 μg/mL of F(ab′) 2 goat anti–human IgM for the indicated time periods. The cell lysates were subsequently separated on a 10% SDS-PAGE gel and analyzed by Western blotting with anti–phospho-BLNK or anti-BLNK mAb. The resulting values are expressed as fold changes in protein expression compared with unstimulated control cells. The values are the mean ± SD of 3 independent experiments (**P < .01 vs controls).
