Signaling pathways regulated by SDF1/CXCR4 in MM. (A) Immunoblotting for pERK and pAKT, demonstrating rapid activation in response to SDF-1 30 nM in a time-dependent fashion at 1, 3, and 5 minutes. (B) Immunoblotting for total CXCR4 demonstrating up-regulation of CXCR4 by SDF-1 (30 and 100 nM for 5-minute incubation) and inhibition by AMD3100 (100 μM for 16-hour incubation), even in the presence of SDF-1 stimulation. (C) Immunoblotting for pPI3K (p85) demonstrating activation in response to SDF-1 in a dose-dependent fashion with maximum activation at 100 nM at 5 minutes. This effect was abrogated by AMD3100 (30-100 μM for 16-hour incubation), confirming that SDF-1 activates PI3K through CXCR4. (D) Immunoblotting for PKC, pAKT, and pERK1/2 in the presence or absence of AMD3100 (30 μM for 16-hour incubation). SDF-1 led to a rapid up-regulation of pPKC, pAKT, and pERK1/2 at 1 minute and 5 minutes. AMD3100 inhibited the expression of pPKC, pAKT, and pERK1/2. (E) Immunoblotting with CXCR4 knockdown MM.1S (lanes 3-4) and mock-infected MM.1S (lanes 1-2), with or without stimulation with 30 nM SDF-1 for 1 minute. CXCR4 knockdown with shRNA led to the inhibition of CXCR4, p-PDK-1, pAKT, and pERK1/2, but not p-p38 MAPK. (F) Immunoblotting for pERK and pAKT in the presence of 50 ng/mL pertussis toxin (PTX) for 90 minutes. SDF-1 (30 nM) induced pERK and pAKT as a control in lane 1. PTX inhibited pERK and pAKT even in the presence of SDF-1, indicating that activation of these pathways by SDF-1 is Gi dependent. (G) Immunoblotting for pAKT and pERK in the presence of the PI3K inhibitor LY294002 (25 μM for 20 minutes) or the MEK inhibitor PD098059 (25 μM for 90 minutes) with or without SDF-1. LY294002 inhibited pAKT even in the presence of SDF-1, whereas PD294002 did not affect AKT activity. LY294002 inhibited pERK1/2, indicating that ERK/MAPK is downstream of PI3K. PD294002 inhibited pERK activity even in the presence of SDF-1.