HGF induces phosphorylation of MET in DLBCL cells and activates the RAS/MAPK and PI3K/PKB pathway. (A) mRNA expression of MET in DLBCL cell lines. After RNA isolation and cDNA synthesis, RT-PCR for MET was performed. β2-Microglobulin was used as housekeeping gene control. (B) MET protein expression in DLBCL cell lines. DLBCL cell lines were analyzed by immunoblotting for the expression of MET. The (weak) expression of MET by OCI-LY-1 cells is clearly demonstrated by means of a 3-times longer exposure (right). Staining with anti-β-actin represents the loading control. (C) HGF induces tyrosine phosphorylation of MET, PKB, FOXO3a, GSK3, and ERK. The DLBCL cells OCI-LY-1, OCI-LY-3, and OCI-LY-10, and MET-transfected Namalwa cells (V3M) were stimulated with HGF for the indicated time periods. Cell lysates were immunoblotted with phosphorylation-specific antibodies against MET, FOXO3a, GSK3, PKB, and ERK. The blots were stripped and restained with antibodies against MET, PKB, and ERK. (D) HGF-induced phosphorylation of FOXO3a and GSK3 requires PI3K activity, whereas phosphorylation of ERK1 and ERK2 is MEK dependent. OCI-LY-3 and OCI-LY-10 cells were pretreated with the PI3K inhibitors wortmannin (WM, 50 μM) or LY294002 (LY, 20 μM), the MEK inhibitor PD98059 (PD, 50 μM), or DMSO (C) for 30 minutes, prior to incubation with HGF (200 ng/mL). Phosphorylation of ERK1 and ERK2, PKB, GSK3, and FOXO3a was determined by immunoblotting with phosphorylation-specific antibodies. The blots were stripped and restained for ERK1 and ERK2, and PKB.