Figure 5

miR-148b, miR-326 and miR-520a affect the ErbB signaling pathway at multiple levels. (A) MCF7 cells were transfected with miR-con, miR-148b, miR-326 and miR-520a (miR-520a-3p), respectively. Three days after transfection, cells were lysed and lysates analyzed by immunoblotting using ErbB3 and PI3K p110α specific antibodies. Tubulin was detected to confirm equal loading. A representative Western blot is shown; the left panels are from the same blot. Signals were quantified by ImageJ and normalized to those of tubulin. Data are shown as the mean ± SEM of three to five independent experiments and analyzed by one way Anova followed by Tukey’s multiple comparison test (* p < 0.05). (B) MCF7 cells were transfected with the indicated miRNAs. Two days post transfection, RNA was extracted and ErbB3 levels were determined by qRT-PCR. Values were normalized to GAPDH. Data are shown as the mean ± SEM of two independent experiments. (C) HEK293T cells were co-transfected with the indicated miRNAs and the different luciferase reporter constructs containing wild-type (WT) ErbB3 mRNA sequences or those lacking the respective miRNA seed regions (mt) (see Methods and Additional file 1: Figure S7). The next day, cells were lysed and luciferase activity measured and normalized to the activity of the co-expressed Renilla reporter. Data correspond to the mean ± SEM of three independent experiments performed with at least triplicate samples. Data analysis: one way Anova followed by Tukey’s multiple comparison test (*p < 0.05, ***p < 0.001). (D) MCF7 cells were transfected with the indicated miRNAs. Three days post transfection, RNA was extracted and SOS1, ADAM17 and RAP1A levels were determined by qRT-PCR. Values were normalized to GAPDH. Data are shown as the mean ± SEM of three independent experiments and analyzed by one way Anova followed by Tukey’s multiple comparison test (*p < 0.05).