GCKR is a downstream target of Bcr-Abl in Bcr-Abl⁺ cell lines. (A) Increased GCKR kinase activity in Bcr-Abl⁺ cell lines. GCKR immunoprecipitates from lysates prepared from 5 × 10⁶ cells of the indicated cell line were subjected to an in vitro kinase assay. The data were normalized to the results with either U937 cells or Jurkat cells. The data shown summarize the results from four experiments and are presented as mean +/− 2 SD. Above the results of the in vitro kinase assay is an immunoblot showing the levels of GCKR in cell lysates prepared from the different cell lines. (B) Bcr-Abl and GCKR associate in K562 cells. Lysates prepared from 1 × 10⁷K562 cells were immunoprecipitated with an antiphosphotyrosine MoAb (PY), a HA MoAb, the GCKR antiserum, preimmune (PI) antiserum, or an Abl polyclonal antiserum as indicated. The immunoprecipitates were subjected to immunoblotting with the indicated antibodies. The GCKR immunoblot (lanes 1 to 3) was stripped and reblotted for Abl (lanes 4 to 6). The Abl immunoblot (lanes 7 to 9) was stripped and reblotted for GCKR (lanes 10 to 12). Bcr-Abl and GCKR were detected in 50 μg of cell lysate (lanes 7 and 10, respectively). Molecular weight markers and the location of Bcr-Abl and GCKR are indicated.