FIG. 1.

Grb10 increases ligand-dependent ubiquitination of the IGF-IR in cells. p6 and p6/Grb10 cells were plated in triplicate onto six-well plates and transiently transfected with the HA-Ub construct. After 24 h, cells were shifted to SFM for an additional 24 h and then stimulated for 10 min with 20 ng of IGF-I/ml in the presence of 20 μM MG132 and 0.4 mM chloroquine to accumulate the ubiquitinated forms. Cell lysates were pooled, and 800 μg was immunoprecipitated (IP) with anti-IGF-IR antibody and blotted with anti-HA antibodies to detect ubiquitinated species (A). This blot was then stripped and reprobed with antiphosphotyrosine antibodies (B) and anti-IGF-IR antibodies to test for the amount of receptor immunoprecipitated (C). Total lysates served as a positive control for HA-Ub transfection (D). Results in panels A to D are representative of three independent experiments. (E) The IGF-IR, but not coprecipitated protein(s), is ubiquitinated in p6/Grb10 cells. After transient transfections as above, lysates of IGF-I-stimulated p6/Grb10 cells were treated with 1% SDS and boiled for 5 min prior to immunoprecipitation with anti-IGF-IR antibodies. After immunoprecipitation, the filter was probed with anti-HA (left) to detect IGF-IR ubiquitination or anti-IGF-IR (center) antibodies (control). Total lysate served as a positive control for HA-Ub transfection (right). (F) IGF-I-induced ubiquitination of the IGF-IR in p6/Grb10 cells is detectable at endogenous levels of ubiquitin. Lysates (2 mg) of IGF-I-stimulated p6/Grb10 cells were immunoprecipitated with anti-IGF-IR antibodies. After immunoprecipitation the filter was probed with antiubiquitin (UB) antibody (left and right) to detect the ubiquitinated IGF-IR. The blot with anti-IGF-IR antibodies (center) is a control. Results in panels E and F are representative of two independent experiments.