FIGURE 4.

CHIP interacts with GHR. A–C, Myc-CHIP was transiently transfected into GHR-expressing U2OS cells (A) or transfected together with wild type GHR or empty vector (EV) in HEK293-TR cells (B and C). A and B, the cell lysates were incubated with biotin-GH or anti-GHR(T), and the protein complexes were isolated on immobilized streptavidin (PD) and protein A beads (IP), respectively, and eluted with SDS sample buffer. C, cells were incubated for 2 h with biotin-GH on ice, and protein complexes from the cell surface were isolated using immobilized streptavidin, eluted with SDS sample buffer. D, cell lysates from Myc-CHIP, Myc-CHIPΔTPR, or empty vector transfected U2OS cells were incubated with biotin-GH or PBS (control), and protein complexes were isolated, washed on streptavidin beads, and analyzed for CHIP on Western blots. E, Myc-CHIP was transiently expressed in U2OS cells. The cells were lysed and incubated with glutathione beads preloaded with GST-GHR270–318 or GST-GHR270–334 fusion proteins. The beads were washed and analyzed for CHIP on a Western blot (WB). TCL, total cell lysate; m, mature GHR; p, precursor GHR. The data in this figure are representative of three independent experiments.