Clustering of FcγRIIa or the γ-chain of FcγRs is sufficient for SHIP phosphorylation.
(A) The expression of CD8 chimeras in stable RAW264.7 transfectants was examined by fluorescence-activating cell sorter (FACS) analysis. The cells were stained with biotinylated F(ab′)2 fragments of OKT8 followed by FITC-conjugated streptavidin and analyzed by FACS. Dotted lines indicate fluorescence of unstained cells. (B,C) The RAW264.7 transfectants were stimulated with biotinylated F(ab′)2 fragments of OKT8 followed by streptavidin. Whole-cell lysates (B) or SHIP immunoprecipitates (C) were separated by SDS-PAGE and blotted with antiphosphotyrosine (anti-pTyr). The filter in C was reprobed with anti–SHIP antibody (lower panel). (D) The expression of CD8 chimeras in stable THP-1 transfectants was examined by FACS analysis using biotinylated F(ab′)2 fragments of OKT8 followed by FITC-conjugated streptavidin. (E,F) THP-1 transfectants were stimulated with biotinylated F(ab′)2fragments of OKT8 followed by streptavidin. Whole-cell lysates (E) or SHIP immunoprecipitates (F) were blotted with anti-pTyr. In panel F, untransfected cells were also stimulated with Fab fragments of IV.3 antibody followed by F(ab′)2 fragments of goat anti–mouse antibody (2 lanes on the farthest right). The membrane was reprobed with anti–SHIP antibody (lower panel).
