Figure 6

Effects of MP470-Erlotinib combination on tyrosine phosphorylation of the HER family and PI3K/Akt. LNCaP (a) and T47D (b) cells were serum starved for 24 hr, pretreated with 10 μM drug as indicated for 2 hr, and then treated with pervanadate (100 μM) for 10 min. Cell extracts were incubated with anti-EGFR, anti-HER2 and anti-HER3 antibodies at 4°C overnight. The immune complexes were enriched by Protein G-Agarose beads and probed by immunoblotting for phosphotyrosine (PY20) and the p85 regularly subunit of PI3K. Western blotting analysis for phosphorylated Akt was performed in T47D cells. (c). SiRNA knockdown of HER2 decreased phosphorylated Akt. LNCaP cells were grown to 70% confluence and treated with non-targeting siRNA (control RNAi) and siRNA against HER2 at a concentration of 100 nmol/L. At 72 hr, cells were harvested to detect HER2, phosphorylated Akt and total Akt by Western blotting. GAPDH was used as a loading control.