Analysis of the effects of p185 Bcr-Abl point mutations on GCKR. (A) Effect of p185 Bcr-Abl point mutants on the immunoprecipitation of GCKR by a phosphotyrosine MoAb. p185 Bcr-Abl wild-type and mutant protein expression vectors (4 μg each) were transfected into HEK 293T cells (2 × 10⁶) along with the HA-GCKR expression vector (2 μg). The presence of p185 Bcr-Abl, p185 Bcr-Abl mutant proteins, and HA-GCKR in antiphosphotyrosine immunoprecipitates (MoAb 4G10) was assessed by immunoblotting with either anti-Abl or anti-HA. The levels of the p185 Bcr-Abl point mutants and HA-GCKR in the cell lysates were also assessed. (B) Effect of p185 Bcr-Abl point mutants on GCKR and SAPK activity. The GCKR and SAPK in vitro kinase assays were performed similar to Fig 1. The fold inductions relative to GCKR alone (control) are shown and were assessed by scanning the autoradiographs and quantified using NIH Image. Immunoblotting showed similar levels of HA-GCKR. The kinase assays were performed five times with similar results.