FIGURE 3.

Ubc13 activity is essential for GHR endocytosis. A, GHR-expressing U2OS cells were transfected with HA-tagged constructs of Ubc13 or Ubc13 mutants (C87A, A98D, C87A, and A98D) and incubated with Cy3-GH for 30 min at 37 °C. The cells were fixed and immune stained with anti-HA and a secondary antibody conjugated with Alexa 488. Localization of Cy3-GH and HA was determined by confocal microscopy. B, CHIP acts specifically on GHR endocytosis. U2OS cells expression both GHR and EGFR were transfected with siRNA specific for CHIP or control siRNA and incubated with Cy3-GH and Alexa 488 EGF for 30 min. The cells were fixed, and the labels were visualized on a LSM510 confocal microscope using Zen software. Bar, 20 μm. The data in this figure are representative of three independent experiments.