Characterization of insulin-like growth factor 1 receptor overexpression, autophosphorylation, signaling transduction and proliferation of MCF7/insulin-like growth factor 1 receptor cells in response to insulin-like growth factor 1. (a) Immunofluorescence showing insulin-like growth factor 1 receptor (IGF-1R) overexpression in MCF7/IGF-1R cells compared to parental MCF7 cells. IGF-1R was probed with mouse monoclonal antibody against IGF-1Rβ (green). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (blue). Original magnification, × 60. (b) IGF-1R autophosphorylation and signal transduction in time course exposure to IGF-1 (100 ng/mL) for 1, 5, 10, 30 and 60 minutes. ERα, estrogen receptor. (c) Proliferative response of MCF7 versus MCF7/IGF-1R cells to IGF-1 stimulation at the dose ranges indicated. Sulforhodamine B (SRB) absorbance values (optical density (OD) 510 nm) are representative of three independent experiments. Data are expressed as means ± SD. (d) IGF-1R autoactivation and signal transduction to IGF-1 exposure at various dose levels (1, 3, 10, 33 and 100 ng/mL) for two hours. Total IGF-1R levels were detected by IGF-1Rβ antibody at 97 kDa. IGF-1R triple tyrosine phosphorylation was determined by using phospho-IGF-1Rβ (Tyr1131) and phospho-IGF-1Rβ (Tyr1135/Tyr1136) rabbit antibodies. Phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated protein kinase B (p-Akt) were determined by using p-ERK1/2 and p-Akt antibodies, respectively. The status of the estrogen receptor (ER) was detected by rabbit anti-ERα antibody. Tubulin was detected as a control for protein loading.