Shc is tyrosine phosphorylated and associated with Grb2 upon FcR stimulation. (A) Antiphosphotyrosine blot of anti-Shc immunoprecipitates. U937IF lysates from resting cells and those stimulated with anti-FcR (A77) and RM antibodies (both Fab′₂ fragments) for different periods were immunoprecipitated with rabbit anti-Shc antibody and immunoblotted for phosphotyrosine. Lane 1 represents immunoprecipitation performed with preimmune antisera of U937IF cells stimulated with anti-FcR and RM for 1 minute. Anti-Shc immunoprecipitates from resting U937IF cells (lane 2) and from U937IF cells stimulated with anti-FcR and RM for 1 minute (lane 3), for 5 minutes (lane 4), for 10 minutes (lane 5), and for 30 minutes (lane 6), respectively. Lane 7 represents a whole cell lysate of U937IF cells stimulated with anti-FcR and RM antibodies for 1 minute. The p145 protein was determined to be SHIP. (B) Anti-Shc (upper panel) and anti-Grb2 (lower panel) immunoblots performed on the same membrane of (A) after stripping with 0.1 mol/L glycine, pH 2.5, at room temperature for 30 minutes. Lanes are identical to those in (A).