Fig. 1

IR/IGF1R ratio is regulated by TRIP-Br1 in breast cancer cells. A Expression levels of TRIP-Br1, IGF1R, and IR were checked in breast normal and cancer cell lines by western blotting. β-actin was used as a loading control. B IR/IGF1R ratio was quantified using ImageJ. C, D Endogenous IR expression was assessed in MEF cells isolated from TRIP-Br1 wild-type (MEF^WT-TRIP-Br1) or knockout mice (MEF^KO-TRIP-Br1), as mentioned in the Materials and Methods (n > 3) (Additional file 3). E, F The IR protein levels from adipocytes tissue collected from TRIP-Br1 wild-type or knockout mice were evaluated by western blotting (n = 3). G, H The TRIP-Br1 and IGF1R expression levels were measured in MEF^WT-TRIP-Br1 or MEF^KO-TRIP-Br1 cells by western blotting. I, J The protein levels of TRIP-Br1 and IGF1R were checked in adipocytes tissue collected from TRIP-Br1 wild-type or knockout mice (n = 3). K The relative IR/IGF1R ratio is shown in MCF7 stable cell lines with TRIP-Br1 wild-type (MCF7^WT-TRIP-Br1) and knock-down (MCF7^KD-TRIP-Br1) cells. L The indicated protein levels were evaluated by western blotting. The expression of IGF1R and IR was co-analyzed using a co-antibody that recognizes both IGF1R and IR. M, N TRIP-Br1 or NEDD4-1 silencing RNA (siTRIP-Br1 and siNEDD4-1) were transfected into indicated cell lines and IGF1R expression was analyzed by using a western blot analysis (n > 3). O The interaction between IGF1R and TRIP-Br1 was determined by using co-immunoprecipitation assay. P, Q The representative images of NEDD4-1 and IGF1R expression were observed using a confocal microscope. The co-localization between NEDD4-1 and IGF1R was measured by counting over 50 cells in ImageJ. Data are presented as the mean ± SD (n > 50). R, S Cells were transfected with siNEDD4-1 in the absence or presence of MG132 (10 μM) for 24 h and subjected to western blotting (n = 3). The quantification results are presented as the mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.005)