The SOCS2 Ubiquitin Ligase Complex Regulates Growth Hormone Receptor Levels
Figure 3
(A) HEK293T cells were transfected with Elongin B/C and Myc-GHR and either an Empty vector, FLAG-SOCS2 or a SOCS2 mutant version. C denotes the control which was tranfected with FLAG-SOCS2 and Elongin B/C but not GHR. Cells were starved for 4 hours in Serum-free media prior to treatment. Next, cells were treated with GH for the times specified in the figure and subsequently lysed as described in the Materials and Methods. Lysates were visualized by western blot. GHR was detected with an anti-Myc antibody. The immature form of the GHR (i) is visible after a short exposure of the film (top panel) whilst the mature form (m) is visible after a longer exposure (2nd panel from the top). (B) Co-Immunoprecipitation of GHR. HEK293T cells were transfected with Elongin B/C, FLAG-SOCS2 or a mutant version of SOCS2 together with Myc-GHR (lanes 1,2 and 7–10) or only FLAG-SOCS2 and Elongins (lanes 5,6) or Myc-GHR alone (lanes 3,4). Cells were starved and treated with GH for 0 (−) or 10 (+) minutes prior to lysis. Lysates were immunoprecipitated as described in the materials and methods and results visualized with western blot. GHR was detected with an anti-Myc antibody. (C) HEK293T cells transfected with either Myc-GHR, FLAG-SOCS2 and Elongin B/C or GHR and Elongin B/C alone were treated with cycloheximide (100 µg/ml) and GH (2 µg/ml) for the times indicated in the figure before lysis. Cell lysates were visualized by western blot. C denotes the control which was transfected with Empty vector instead of GHR. GHR was detected with an anti-GHR antibody.