Expression of functional TrkB by MM: intracellular signaling in response to BDNF. (A-B) BDNF triggers phosphorylation of Akt and MAPK by HMCL. HMCLs were serum starved overnight, exposed to ligand for 5 minutes at 37°C, and lysed. Whole-cell lysates were subjected to SDS-PAGE, blotted, and probed with antibodies specific to Akt, phosphoAkt, MAPK, or phosphoMAPK. Blots were developed with ECL, and the resulting images were scanned and analyzed using Scion Image for Windows (Frederick, MD). The density of each phosphospecific band was corrected for variance in loading, using the density of the corresponding nonphosphorylated band. The fold induction was then calculated as the ratio of densities between stimulated and unstimulated phosphorylated protein. (A) BDNF triggers phosphorylation of Akt in HMCL. The ability of NGF (100 ng/mL), BDNF (50 ng/mL), or IL-6 (100 ng/mL) to activate PI3K/Akt signaling in HMCL was assessed using antibodies specific to Akt and phosphoAkt. (B) BDNF triggers phosphorylation of MAPK in HMCL. Antibodies specific to MAPK and phosphoMAPK p42/44 were used to assess activation of Ras/Raf/MEK signaling by BDNF (50 ng/mL) in HMCLs OPM1, U266, and KMS11. (C-D) BDNF triggers phosphorylation of Akt and CREB in primary MM cells. BM mononuclear cells were stimulated for 10 minutes at 37°C with BDNF (50 ng/mL), spun onto microscope slides, and stained for CD138 (FITC) and phosphoAkt (rhodamine), or CD138 (FITC) and phosphoCREB (rhodamine). (C) Akt phosphorylation by primary MM. CD138⁺ cells (green) stain for cytoplasmic phosphoAkt (red) after stimulation with BDNF (merged image is yellow). (D) CREB phosphorylation by primary MM. CD138⁺ cells (green) show nuclear localization of pCREB (red) after stimulation with BDNF.