Figure 2. Analysis of IGF1R, IR, and IGF1R/IR HR levels in cancer cells sensitive to anti-IGF1R antibody.

A) Immunoblot analysis of total IGF1Rβ and IRβ protein levels. Two types of gastric and hepatocellular carcinoma cells were harvested 24 h after plating and immunoblotting with anti-IGF1Rβ antibody, anti-IRβ antibody, and anti-α tubulin antibody was performed. For both types of cells, representative blots from three independent experiments are shown. B) Analysis of the presence of IGF1R/IR heterodimeric receptor (HRs) using immunoprecipitation. Total cellular proteins (1 mg) from cells were used for immunoprecipitation with anti-IGF1R antibody, separated by SDS-PAGE at constant voltage (80 V), and Western blotted with an anti-IRβ antibody. The blot was then stripped and reprobed with anti-IGF1Rβ antibody C) Quantitative analysis of IGF1R homodimer, IR homodimer, and IGF1R/IR heterodimer levels using an ELISA. Lysis buffer (100 µL) containing equal amount of proteins (50 µg/well) from 11 cancer cell lines including MCF7 cells (positive control) were plated on anti-IGF1R antibody-coated wells and detected with an anti-IR detection antibody. Anti-IR antibody-coated wells, IR protein standards, and the anti-IR detection antibody were used as standards in the heterodimeric receptor ELISA. Absorbance was measured at 450 nm. Values are expressed as the mean±SEM nanograms of receptor protein per 50 µg total protein. Cell lines are listed according to their sensitivity to figitumumab. Bars = ±SE. D) Effect of figitumumab on IGF1- mediated IGF1R/IR HRs. Cells were serum-starved for 24 h and then treated with figitumumab, IGF1, or left untreated. SNU719 cells were incubated with figitumumab (10 µg/mL) for 1 h at 37°C followed by stimulation with IGF1 (100 ng/mL) for 30 min. Immunoprecipitation was performed with an anti-IGF1R antibody and Western blotted. Input = total cell lysate without IP.