Fig. 3. Insulin induces interaction between EGFR and IGF-1R. Cells were stimulated with 10 μg/ml insulin for 1 h after treatment in serum-free media containing 30 μg/mL cycloheximide for 8 h. (A) Schematic figure showing the principle of proximal ligation assay. (B) Proximity ligation assay was performed according to the manufacturer’s instructions and cell images were obtained using fluorescence microscopy (Applied Precision Delta Vision, 60× oil objective with a 1.35 numerical aperture). Red dots indicate signals resulting from the interaction between EGFR and IGF-1R. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 ㎛. Ins, insulin. (C) Cell images were analyzed using the Duolink^® ImageTool software and statistical analyses were performed using GraphPad software. Error bars in the graph represent SD (n = 3; Student’s t-test, *P ＜ 0.05). (D) Immunoblot analysis was performed using anti-EGFR and anti-p-EGFR on MDA-MB-436 and -468 cell lines, respectively.