Effect of VEGF on the Raf-MEK-ERK pathway.
(A) MM.1S cells were starved overnight in RPMI 1640 with 1% FBS and for 3 hours in RPMI 1640 with no FBS. After stimulation of MM.1S cells with 100 g/mL VEGF165 for 5 minutes, Flt-1 immunoprecipitates (IP) from whole cell lysates were analyzed by Western blotting with the use of antisera against phospho-tyrosine residues. Equal loading was confirmed by immunoblotting with antisera directed against Flt-1, and whole cell lysate of VEGF-stimulated HUVECs was similary immunoblotted as a positive control. Nonspecific protein binding and detection were excluded by incubating protein A–Sepharose (PAS) beads with lysis-buffer and Flt-1 antibody only (control). (B) MM.1S, patient MM, and patient PCL cells, pretreated as described in panel A, were stimulated with 100 ng/mL VEGF165 for 5 minutes, 15 minutes, and 45 minutes. Whole cell lysates (40 μg) were analyzed by Western blotting with antisera against phospho-ERK (pERK). Immunoblotting for ERK-2 confirmed equal protein loading. (C) Densitometry was used to quantitate pERK activity in panel B. The data shown are representative of 3 separate experiments.
