Full-length Grb4 interacts with Bcv-Abl.

(A) Grb4 interacts with Bcr-Abl and Abl in a chronic myeloid leukemia cell line. Cell lysates from 3 × 10⁷ K562 cells were incubated with 5 μg of GST or GST-Grb4 (full length), and the bound fraction was resolved on SDS-PAGE and then blotted with an anti-Abl antibody (8E9). Bands indicated as Bcr-Abl and Abl correspond to 210 kd and 145 kd, respectively. (B) Full-length Grb4 interacts with Bcr-Abl in vivo. Then, 1 × 10⁶ 293 cells were transfected with KD Bcr-Abl (BA KD), WT Bcr-Abl (BA WT), or ts Bcr-Abl (BA TS) together with Grb4. Cells transfected with the ts mutant were shifted to 32° or 39° 8 hours before harvest. A coimmunoprecipitation experiment was performed with the use of a rabbit antimouse antibody (RαM) as control (left panels) or an anti-Xpress antibody to detect tagged Grb4 (right panels). Bound fractions were resolved on SDS-PAGE and immunoblotted with an anti-Abl antibody (8E9) (upper panels) or a phosphotyrosine antibody (4G10) (lower panels). In the upper panel, lanes 1 to 4 and 7 to 8 were exposed 3 times longer than lanes 5 and 6. Levels of Grb4 immunoprecipitated in all lanes were equal (not shown).