CXCR4 and VLA-4 cointeract and regulate downstream P13K and ERK/MAPK pathways. (A) Coimmunoprecipitation using anti–VLA-4 antibody and blotting for CXCR4 antibody, showing that SDF-1 30 nm for 3 minutes induces coimmunoprecipitation of CXCR4 with VLA-4 indicating direct interaction of these 2 receptors. (B) Immunoblotting of BCWM.1 stimulated with SDF-1 30 nM for 1, 2, 3, and 5 minutes. SDF-1 induced activation of pERK and pAkt within 1 and 3 minutes, whereas PKC activation occurred at 3 to 5 minutes. Anti–β-actin was used as a loading control. (C) Immunoblotting of BCWM.1 with SDF-1 30 nM for 3 minutes and in the presence of AMD3100 20 μM (pretreated for 2 hours followed by SDF-1 activation at the last 3 minutes). AMD3100 inhibited pERK, pAkt, and pPKC activation, even in the presence of SDF-1. (D) Immunoblotting for pERK and pAkt using the mock-infected or CXCR4-knockdown BCWM.1 cell line showing that SDF-1 30 nM for 3 minutes does not significantly activate pERK and pAkt in the CXCR4-knockdown cell line. (E) Immunoblotting with BCWM.1 showing activation of pERK and pAkt by SDF-1 30 nM for 3 minutes, whereas the MEK inhibitor PD098059 20 μM (for 20 minutes) inhibited pERK activation but not pAkt, and the PI3K inhibitor LY294002 (for 15 minutes) 25 μM inhibited pAkt and pERK activation, in the presence of SDF-1 30 nM for 3 minutes. (F) Migration assay of BCWM.1 in response to 30 nM SDF-1 using PD098059 20 μM for 20-minute pretreatment, or LY294002 25 μM for 15-minute pretreatment, showing that PD098059 and LY294002 inhibit migration of BCWM.1 in response to SDF-1. Data show an average of 3 independent experiments (*P = .01, **P = .05).