The β₁-integrin/hERG1/CXCR4 complex regulates MSC-induced signaling. (A) Western blot of 697 cells treated for 30 minutes with BSA (250 μg/mL), SDF-1α (50 ng/mL), the β₁-integrin-activating antibody TS2/16 (20 μg/mL), or cultured on MSCs with or without the anti–β₁-integrin-blocking antibody. Total cell lysates were immunoprecipitated with a polyclonal ILK antibody, and a kinase assay was performed with the synthetic GSK3 protein as a substrate. Phosphorylated substrates were detected by Western blot with the anti–pGSK-3 Ser^9/21 antibody. Densitometric measurements relative to the blot reported are shown on the bottom. Experiments reported in the panel are representative of at least 4 independent experiments. (B) Western blot of pERK1/2 (left panel) and pAkt (right panel) in 697 cells after coculture for 30 or 60 minutes with MSCs in the absence or presence of anti–β₁-integrin-blocking antibody. The membrane was then reprobed with an anti-ERK1/2 or anti-Akt antibody and anti-tubulin antibody. Densitometric measurements relative to the blot reported in the panel are shown on the bottom. Experiments shown are representative of at least 4 independent experiments. (C) 697 cells were cultured on MSCs for 30 minutes with or without an hERG1 blocker (WAY or E4031, both at 20μM). Cell lysates were immunoprecipitated and processed as in panel A. Densitometric measurements relative to the blot reported are shown on the bottom. Experiments shown are representative of at least 4 independent experiments. (D) Western blot of the levels of pERK1/2 and pAkt in 697 cells after coculture with MSCs for 30 minutes, 60 minutes, or 24 hours with or without the hERG1 inhibitor E4031 (20μM). Total levels of ERK1/2 and Akt and tubulin proteins are shown. Densitometric measurements relative to the blot reported are shown on the bottom (white, −E4031; gray, +E4031). Experiments shown are averaged in panel E and are representative of 5 independent experiments. (E) pERK1/2 (top panel) and pAkt (bottom panel) in leukemic cells cultured on MSCs in the absence (dashed line) or in the presence of hERG1 inhibitors (solid black line) over 24 hours; densitometric measurements shown represent the mean of 5 different experiments. *P < .05. (F) Western blot of pERK and pAkt levels in primary clinical samples of BCP-ALL after coculture with MSCs for 30 or 60 minutes in the absence or presence of the hERG1 inhibitor E4031 (20μM). Total levels of ERK1/2 and Akt proteins and tubulin are shown in the bottom panels.