?(Fig

?(Fig.11 BCR, B cell antigen receptor; CD40L, CD40 ligand; ECM, extracellular matrix; FDC, follicular dendritic cells; GC, germinal center; HGF/SF, hepatocyte growth factor/scatter factor; PKC, protein kinase C; SAC, Cowans strain; VCAM-1, vascular cell adhesion molecule 1.. were removed by using a MACS magnetic cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany) using anti-CD19. The T cell portion was >98% real as determined by FACS? analysis. Stromal Cell Isolation and Culturing. Tonsillar stromal cells were isolated as explained (46). The cells were cultured in 100-mm petri dishes (Costar, Cambridge, MA) made up of 10% FCS/RPMI 1640. After 4 d nonadherend cells were removed. FDC Isolation and Culturing. FDC were isolated as explained (29). The cells were cultured in Iscove’s medium made up of 10% Fetal Clone I serum (HyClone Laboratories, Logan, UT). These FDC-enriched cell cultures contained 10C15% DRC-1Cpositive cells. Transfections. c-Met transfected Namalwa cells (NamcDNA (a gift from G. Hartmann and E. Gherardi, University or college of Cambridge, Cambridge, UK). After 2 d in culture, transfectants were selected in culture medium made up of 250 g/ml hygromicin (or EB4B cells were incubated in serum-free RPMI 1640 in the presence or absence of 200 ng/ml HGF/SF (R&D Systems). After 5 min at 37C the cells were solubilized in ice-cold 2 lysis buffer made up of 20 mM Tris-HCl (pH 8), 250 mM NaCl, 20% glycerol, 2% NP-40, 20 g/ml aprotinin (at 4C for 20 min after which the supernatant was precleared with protein ACSepharose CL-4B (Biotech) for 45 CGS 35066 min at 4C. c-Met was precipitated with rabbit antiCc-Met coupled to protein ACSepharose at 4C for at least 2 h. The immune complexes were washed with lysis buffer and diluted in Laemmli sample buffer containing final concentrations of 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 100 mM 2-mercaptoethanol (BioRad Laboratories), and 0.001% bromophenol blue. After boiling for 5 min, HNPCC1 the samples were subjected to 8% SDS-PAGE. Western blotting was performed as explained previously (48). For analysis of c-Met in total cell lysates, cells were lysed in 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% NP-40, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM sodium orthovandate, 2 mM EDTA, and 5 mM NaF for 1 h at 4C. After centrifugation at 1 104 and 4C for 20 min, the supernatant was diluted in Laemmli sample buffer, boiled for 5 min and subjected to 8% SDS-PAGE. Western blotting was performed as explained previously (48). FACS? Analysis. Expression of c-Met on tonsillar B cell subpopulations was analyzed using a triple staining technique (47). Staining was measured by using a FACSCalibur? circulation cytometer CGS 35066 (Biotech). PCR was performed with Taq DNA Polymerase (Gibco BRL/Life Technologies), 200 M dNTPs (Biotech) and 1.5 CGS 35066 mM MgCl2 in 1 PCR Buffer (both Gibco BRL/ Life Technologies). Primers used were HGF-1 (5-CGACAGTGTTTCCCTTCTCG-3) in combination with HGF-3 (5-GGTGGGTGCAGACACAC-3), or 52M (5-ATCCAG CGTACTCCAAAGATT-3) in combination with 32M (5-CATGTCTCGATCCCACTTAAC-3). PCR was started with a 5 min denaturation step at 95C, after which amplification was performed in 35 cycles of denaturation at 95C for 30 s, annealing at 60C for 1 min and elongation at 72C for 2 min. After a final elongation step for 10 min at 72C, samples were cooled on ice and analysed by electrophoresis in a 1.5% agarose TBE gel containing ethidium bromide. Results The c-Met Receptor Tyrosine Kinase Is usually Expressed by Activated Human Tonsillar B Cells as well as by Several B Cell Lines. Expression of c-Met by human tonsillar B cells and by a panel of B cell lines was assessed by Western blotting and by FACS? analysis. On Western blot, c-Met expression was hardly detectable in freshly isolated tonsillar B cells, but we observed a strong induction of c-Met (and the c-Met precursor [pre c-Met]) upon activation with the phorbolester PMA (Fig. ?(Fig.11 are c-MetCtransfected Namalwa cells. In both and the Western blot of the cell lysates was stained with antiCc-Met. The c-Met precursor ((B cells resulted in an enhanced tyrosine phosphorylation of c-Met. This indicates that this HGF/SFCc-Met pathway on.