Grb4 encodes for a 47-kd protein.
(A) We lysed 1 × 107 293 cells overexpressing Xpress-tagged Grb4 and performed an anti-Xpress immunoprecipitation. Bound proteins were resolved with the use of SDS-PAGE, and an anti-Grb4 Western blot was performed with the use of a polyclonal rabbit antiserum (left panel) or an anti-Xpress antibody (right panel). Blots were visualized by means of the ECL detection system. (B) N-terminal EYFP fusion constructs of Grb4 and Nck were transiently expressed in Cos7 cells. After 3 days, 1 × 107 cells were lysed and separated by SDS-PAGE and probed with an anti-EGFP antibody (Clontech) (left panel) and the anti-Grb4 serum (right panel). Bands were visualized by means of the ECL system. (C) Then, 1 × 107 293 (overexpressing Xpress-tagged Grb4), Jurkat, Mo7e, Mo7e expressing p210/Bcr-Abl, and K562 cells were lysed and resolved on SDS-PAGE, and Western blotting was performed with the Grb4 antibody. Native Grb4 migrated at 47 kd and is indicated by the arrow. Tagged Grb4 migrates slightly more slowly (lane 1). (D) Then, 2 × 107 293 cells overexpressing Grb4 were lysed, and an immunoprecipitation (Grb4 Ip) followed by immunoblotting was performed with the use of the rabbit polyclonal Grb4 antibody or a control rabbit antibody.
