Fig. 3

RAGE is involved in the activation of Ins/IR signals. The activation of IR (Y1135/1136), IRS1 (Y612) and AKT (S473) observed in MCF-7 (A–C) and ZR75 (E–G) cells treated with vehicle (−) or Ins (20 nM, 15 min) is abrogated by silencing RAGE. The up-regulation of CD1 observed in MCF-7 (D) and ZR75 H cells treated with vehicle (−) or Ins (20 nM, 4 h) is abrogated by silencing RAGE. Total proteins and β-actin serve as loading control. Co-immunoprecipitation of IR with RAGE in MCF-7 cells overexpressing RAGE (MCF7-RAGE) treated with Ins, as indicated (I). FPS-ZM1 (10 μM, 24 h) prevents the co-immunoprecipitation of IR with RAGE in cells stimulated with Ins (20 nM, 5 min) (J). Total lysates (input) are evaluated as control. In situ Proximity ligation assay in MCF7-RAGE cells treated with vehicle (−) or Ins (20 nM, 5 min). Red fluorescence indicates the membrane proximity of IR and RAGE (< 30–40 nm). Nuclei are stained by DAPI (blue fluorescence) (K). Side bar graph indicates the number of dots/cell. Data shown are the mean ± SEM of at least two independent experiments. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001