Identification of the Ptyr 155 with anti-SHIP2 antibodies. (A) Proteins precipitated from lysates of K562 cells (10 × 106) by anti-SHIP2 antibodies were separated by SDS-PAGE and transferred to Immobilon. Before immunoprecipitation, antibodies were incubated for 20 minutes with the indicated peptide: (−) no peptide; (+) antigenic peptide at 20 μmol/L and 100 μmol/L; and (unr) unrelated peptide at 20 μmol/L. Blot was probed with the anti-Ptyr MoAb, 4G10, and reprobed with anti-SHIP2 antibodies. Molecular weights (in kilodaltons) are indicated on the right side of each panel. (B) NP-40 lysates of K562 cells (10 × 106) were immunoprecipitated with rabbit antibodies to SHC or to mouse IgG (RIgG). Immunoprecipitates were separated by SDS-PAGE, transferred to Immobilon, and immunoblotted with 4G10 and reprobed with anti-SHIP2 antibodies.
