FIGURE 2.

GHR endocytosis requires Ubc13 and CHIP. A, U2OS cells expressing GHR were treated with Ubc13, CHIP, or control siRNA for 3 days; incubated with Cy3-GH and Alexa 488-Tf for 30 min; and fixed. Localization of Cy3-GH and transferrin was determined with confocal microscopy. Bars, 20 μm. B, the cells were treated with CHIP or control siRNA for 2 days. The cell lysates were boiled in sample buffer and analyzed using Western blot and immunostaining with anti-GHR(B), anti-CHIP, and anti-actin. C, cells were labeled for 15 min in the presence of [³⁵S]methionine and then chased for 15 min. After lysis, GHR was immunoprecipitated using anti-GHR(B) and analyzed on a Storm imaging system after electrophoresis. TCL, total cell lysate. D, after silencing for 3 days, initial endocytosis rates were measured 5, 10, or 15 min after a 2-h incubation with 180 ng/ml ¹²⁵I-GH on ice. The amount of intracellular radioactivity was measured and expressed as a percentage of total radio activity. All of the data in this figure are representative of four independent experiments. *, p < 0,001 compared with control.