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. 2017 Oct 31;11(12):1788–1805. doi: 10.1002/1878-0261.12145

Figure 2.

Figure 2

Action of dasatinib on the NRG–HER signaling system in MCF7 cells. (A) Western blotting analysis showing the time‐course effect of dasatinib on the phosphorylation of HER receptors and downstream proteins involved in NRG signaling. In addition, the effect of dasatinib in NRG‐induced MMP13 upregulation was detected. Levels of GAPDH were used as a loading control. (B) Graphical representation of the quantification of HER2 and HER3 phosphorylation after NRG stimulation in MCF7 cells pretreated or not with dasatinib. Data were relativized to maximal phosphorylation in untreated cells and are expressed as the mean ± SD of three independent experiments performed as in (A). (C) Time‐course effect of dasatinib on the interaction of SHC with HER2 and HER3 after NRG stimulation by western blotting. Protein–protein interaction was analyzed by co‐immunoprecipitation. GAPDH levels were used as a loading control. (D) Effect of dasatinib on the time course of RAS activation induced by NRG analyzed by western blotting. A RAS‐GTP pull‐down assay was performed to measure the levels of activation of RAS. Levels of total RAS were used as a loading control. (E) Analysis of the effect of 1 μm trametinib or 10 μm BIX02189 on the regulation of MMP13, pERK5TEY, and pERK1/2 levels after NRG stimulation (15 min for pERK5 TEY and pERK1/2 or 4 h for MMP13) by western blotting. The asterisk indicates the heavy chain band of the antibody used for MMP13 immunoprecipitation. Levels of GAPDH were used as a loading control. Data information: Experiments were repeated three times with similar results. Representative results of all the findings are shown.