Figure 8

Effects of PC-PLC inhibition on EGFR and HER3 internalization, overall receptors' contents and heterodimer formation with HER2. (a) CLSM analyses of fixed SKBr3 cells after exposure to D609 for different time intervals and stained with either α-HER3 (upper panels) or α-EGFR antibodies (lower panels). Nuclei were stained with DAPI (blue). Scale bars, 20 μm. (b) Immunoblotting (IB) and immunoprecipitation (IP) experiments on SKBr3 cells, incubated with D609 for 24 hours, to detect overall HER3 and EGFR contents (left panels) and their levels of heterodimerization with HER2 (right). Central panels show the efficiency of the α-HER3 and α-EGFR antibodies in immunoprecipitating the respective target receptors. ET CTR, total extract of control cells; ET D609, total extract of D609-treated cells; CTR IgG, control IgG of α-HER3 or α-EGFR antibodies; IP CTR, immunoprecipitation from control cells; IP D609, immunoprecipitation from D609-treated cells. Representative examples of two independent experiments are shown.