Figure 4

MP470-Erlotinib combination markedly reduces protein kinase activity. (a-b). LNCaP (a) and NIH3T3 (b) cells were serum starved for 24 hr, pretreated with drugs as indicated for 2 hr, and then treated with pervanadate (100 μM) for 10 min. Whole cell extracts were analyzed by immunoblotting for phosphorylated tyrosine kinases, phosphorylated Akt (Ser473), phosphorylated ERK1/2 (Thr202/Tyr204), and total Akt. (c). LNCaP cells were serum-starved for 24 hr, pretreated with DMSO, 10 μM of MP470 or MP470-Erlotinib, and then stimulated by pervanadate for 10 min. For immunoprecipitation (IP) assays, whole cell extracts (WCE) containing equal amounts of protein were incubated with anti-phosphotyrosine (PY20) antibodies overnight at 4°C. Immune complexes were enriched by Protein G-Agarose beads and probed by Western blotting for the p85 subunit of PI3K.