GCKR and Bcr-Abl associate in vivo. (A) GCKR and Bcr-Abl coimmunoprecipitate. HEK 293T cells were transfected with constructs expressing HA-GCKR and either Bcr-Abl (lanes 1, 3 to 10), or v-Abl (lane 2). Cell lysates (2 × 106 cells) prepared using a digitonin lysis (lanes 1 to 4) or a Triton X-100 lysis buffer (lanes 5 to 10) were immunoprecipitated with anti-HA, anti-Abl, or a control antibody as indicated underneath. The immunoprecipitates or cell lysates were analyzed by immunoblotting with anti-HA (lanes 1 to 7) or anti-Abl (lanes 8 to 10) antibodies. The signal was detected by ECL. (B) Mapping of the region in GCKR required for interaction with Bcr-Abl. Lysates from HEK 293T cells (2 × 106) transfected with constructs expressing HA-tagged GCKR deletion constructs along with Bcr-Abl were immunoprecipitated with anti-Abl or control antibody. The immunoprecipitates and lysates were analyzed by immunoblotting with an anti-HA antibody. The signal was detected by ECL. A nonspecific doublet in the range of 65 kD is present in each of the lysates. The GCKR truncation mutants migrated with their expected molecular masses and were visualized in the cell lysates.
