Effect of GW654652 on Flt-1 phosphorylation and VEGF-triggered activation of downstream signaling molecules. (A) MM.1S cells were starved overnight in RPMI 1640 with 1% FBS and for 3 hours in RPMI 1640 with no FBS. After pretreatment with GW654652 (1 hour; 10 μg/mL) or dimethyl sulfoxide (DMSO), MM.1S cells were stimulated with 100 ng/mL VEGF for 1 and 5 minutes. Flt-1 immunoprecipitates (IPs) from whole cell lysates were analyzed by Western blotting using antisera against phosphotyrosine residues. Equal loading was confirmed by immunoblotting with antisera directed against Flt-1. Nonspecific protein binding and detection were excluded by incubating protein A-Sepharose (PAS) beads with lysis buffer and Flt-1 antibody only (C, control). (B) MM.1S cells pretreated as in panel A were stimulated with 100 ng/mL VEGF in the presence and absence of GW654652 for 1, 5, and 30 minutes. Whole-cell lysates (60 μg) were analyzed by Western blotting using antisera against phospho-ERK (pERK) and phospho-AKT-1 (pAKT1). Immunoblotting for ERK-2 confirmed equal protein loading.