SHIP binds directly to the ITAM of FcγRIIa in vitro and in vivo.
(A) Sequences of peptides used are shown and described previously.29 (B) THP-1 cells were stimulated with Fab fragments of IV.3 antibody followed by F(ab′)2 fragments of goat anti–mouse antibody. The lysates were incubated with biotinylated peptides indicated and purified by Neutravidin beads. The precipitates were resolved by SDS-PAGE, and blotted with anti–SHIP antibody. (C) The recombinant GST-SH2-SHIP fusion protein (right 5 lanes) or GST protein (left 5 lanes) was incubated with biotinylated peptides indicated, precipitated with Neutravidin beads, resolved by SDS-PAGE, and blotted with anti–GST antibody. The phosphorylated peptides were pretreated with calf intestinal alkaline phosphatase (CIAP) before the incubation with recombinant proteins as indicated. The position of GST-SH2-SHIP was indicated at right. (D) THP-1 transfectants were stimulated with biotinylated F(ab′)2 fragments of OKT8 followed by streptavidin and lysates were immunoprecipitated with anti-myc. The immunoprecipitates (ip) were separated by SDS-PAGE and probed with antibody to SHIP (upper panel) and reprobed with anti-myc (lower panel).
