FIG. 2.

Grb10 is involved in regulating IGF-IR stability and internalization. p6 and p6/Grb10 were serum starved in serum-free Met-Cys-free medium, pulsed with [³⁵S]Met-Cys for 1 h, and chased in SFM supplemented with 20 ng of IGF-I/ml for 0, 2, 4, and 8 h. Cells were then lysed, and 300 μg of protein was immunoprecipitated with anti-IGF-IR antibodies. (A) Immunocomplexes were washed, resolved by SDS-PAGE, dried, and exposed to X-ray film. The IGF-IR is newly synthesized as a proreceptor and then processed into α and β subunits. Results are representative of three independent experiments. (B) Quantitation of IGF-IR using densitometric analysis. The graph was generated with the ImageQuant program. (C) Decreased level of cell surface IGF-IR in p6/Grb10 cells. The level of cell surface IGF-IR in p6 and p6/Grb10 cells was determined by ELISA at different times of IGF-I stimulation, as described in Materials and Methods. IGF-I was used at 200 ng/ml to ensure a saturating concentration of the ligand. The data are the averages of three independent experiments.