The β1/hERG1/CXCR4 complex is assembled on the plasma membrane of leukemic cells cultured on MSCs. (A) Coimmunoprecipitation (IP) of β1-integrin, hERG1, and CXCR4 from the REH, RS4;11, and 697 ALL cell lines. Left, cell lysates were immunoprecipitated with the anti–β1-integrin antibody TS2/16 and blots were probed with the anti-pan hERG1 antibody (C54), the anti-CXCR4 antibody, and the anti-β1 antibody. Right, cell lysates were immunoprecipitated with the anti-CXCR4 antibody and blots were probed with the anti-pan hERG1 antibody (C54), the anti-β1 antibody, and the anti-CXCR4 antibody. Experiments shown are representative of at least 6 independent experiments. (B) Effect of stimulation with SDF-1α, β1-integrin, and MSCs on CXCR4/β1/hERG1 complex formation. Leukemic cells were treated for 30 minutes with bovine serum albumin (BSA; 250 μg/mL), SDF-1α (50 ng/mL), the β1-integrin-activating antibody TS2/16 (20 μg/mL; left panel), or were cultured on MSCs; controls were cells treated with BSA (250 μg/mL) for 30 minutes. Experiments shown are representative of at least 4 independent experiments. (C) The 697 cell line was cultured on MSCs for 30 minutes in the absence or presence of the anti–β1-integrin-blocking antibody. Proteins were extracted and immunoprecipitated with the anti-CXCR4 antibody and the blot was sequentially labeled with anti-pan hERG1 (C54), anti–β1-integrin, or anti-CXCR4 antibody. Experiments shown are representative of at least 6 independent experiments. (D) Coimmunoprecipitation of CXCR4, hERG1, and β1-integrin from 3 primary clinical samples. Experiments were performed as described in panel A and shown are representatives of at least 4 independent experiments.
