BCR-ABL1 profoundly inhibits chemotactic responses to SDF-1 and alters SDF-1–induced signal transduction while not decreasing the expression levels of the CXCR4 receptor. (A) A total of 3 polyclonal M-07e cell populations, one control (Ctr; transfected with empty vector) and 2 expressing different amounts of p210–BCR-ABL1, were generated through infection with retroviral vectors (Document S1). Western blotting and kinase assays showed an approximately 5-fold difference in BCR-ABL1 protein and function, respectively, between a high p210 BCR-ABL and low p210 BCR-ABL cell population. Total-cell lysate from M-07e-control vector cells was used as negative control; lysates from K562 cells were used as positive control. (B) CXCR4 surface and total cellular expression in control and BCR-ABL1⁺ M-07e cells were evaluated by FACS (top panel; Document S1). The expression levels of p210 BCR-ABL in these cells were determined by Western blot using c-ABL antibody. The blot was stripped and reprobed with an anti-CXCR4 antibody and then with an anti-GAPDH antibody. CXCR4 membrane and CXCR4 total cellular expression was determined by FACS in normal CD34⁺ cells obtained from healthy individuals and BCR-ABL⁺ CD34⁺ cells from patients with CML in blast crisis (bottom panel). The result shown is representative of 3 independent experiments with 3 different cell donors. The percentage of CXCR4 surface-positive cells was 30.2% plus or minus 7.6% and 27.1% plus or minus 18.9%, and the percentage of CXCR4 total-positive cells was 65.4% plus or minus 15.1% and 48.6% plus or minus 14.1% (mean ± SEM), in normal CD34⁺ cells and CML blast crisis CD34⁺ cells, respectively. (C) Chemotaxis assays in SDF-1–stimulated control M-07e and BCR-ABL1⁺ M-07e cells (Document S1). Values in chemotaxis assays are means plus or minus SEM (n = 4). (D) Chemotaxis assay in SDF-1–stimulated normal CD34⁺ cells and CML blast crisis CD34⁺ cells. Values in chemotaxis assays are means plus or minus SEM (n = 3); *P < .05. (E) Time course of Erk activation after SDF-1 stimulation (100 ng/mL for 5 and 10 minutes) in M-07e cells transfected with control (Ctr) or p210-BCR-ABL (p210) vector was measured by phosphor-Erk (p-Erk) Western blotting (top). The membrane was stripped and reprobed with anti–total Erk (t-Erk; bottom).