Association of Syk and ZAP-70 with the FcεRIγ subunit after receptor clustering in U937 cells. (a) Lysates from unstimulated (−) or FcγRI-stimulated (+) U937, U937/LN, or U937/ZAP-70 cells were immunoprecipitated with an anti-FcεRIγ polyclonal rabbit antibody (5297), separated on a 15% polyacrylamide gel, and analyzed by immunoblotting with an antiphosphotyrosine antibody. The positions of Syk and the FcεRIγ receptor subunit are indicated by arrows. The heavily reactive bands observed at approximately 50 kD and 25 kD are the heavy and light chains of the immunoprecipitating antibody, respectively. (b) The blot was stripped and reprobed with the anti-FcεRIγ polyclonal rabbit antibody (5297). (c, d, and e) Lysates treated as in (a) were separated on a 7.5% polyacrylamide gel and immunoblotted with either a monoclonal antiphosphotyrosine (c), polyclonal rabbit anti-Syk (d), or monoclonal anti–ZAP-70 (e) antibody. These data are representative of results obtained in six independent experiments.