Fig. 4

Downregulation of the HER2 signaling pathway in vitro. a Treatment with BEZ235 in combination with trastuzumab led to complete inhibition of p-AKT and p-S6, with partial inhibition of p-ERK. b p-ERK, p-AKT, and p-S6 were not further inhibited by trastuzumab combined with AZD6244 or Everolimus compared to trastuzumab, AZD6244, and Everolimus monotherapy. The levels of p-AKT, p-ERK, and p-S6 were analyzed by western blotting using specific monoclonal antibodies. β-Actin was used as a loading control. The phosphorylation levels of AKT, ERK, and S6 were quantified by using the software Image J. The data are expressed as the Mean ± S.D of three independent experiments. *P <0.01 by one-way ANOVA or unpaired two-tailed t-test. Cropped blots were used because the same experimental conditions were used for running all the gels