Syk is a major target of FcαR-induced PTK activity. THP-1 cells were activated at 37°C for 5 minutes by the addition of 5 μg/mL Mo2 (lanes 1 and 2), My43 cell culture supernatant 1:2 (lane 3), and RPMI 1640 cell culture medium only (lane 4). Lysates from 1.5 × 107 cells were immunoprecipitated with a rabbit polyclonal anti-Syk antibody (lanes 2 through 4) or control rabbit polyclonal IgG (lane 1). Two thirds of each immunoprecipitate were separated by 8% to 12% SDS-PAGE, transferred to NC sheets, and immunoblotted with an antiphosphotyrosine antibody (A). The immunoblot was afterwards stripped and reprobed with Syk-specific antibody (B). One third of each immunoprecipitate was used for in vitro kinase assay, as described in the Materials and Methods; separated by 8% to 12% SDS-PAGE; and autophosphorylated Syk kinase was detected on dried gels using autoradiography (C).
