SCF(βTrCP) and Clathrin act downstream of Jak2.

<p>A. GHR-expressing Hek293 cells were either silenced for GFP (negative control), βTrCP or clathrin, and then treated for 15 min with GH at 37°C as indicated. GHR immunoprecipitations were analysed for ubiquitination. In the lower part of the figure the efficiency of silencing is visualised using anti-βTrCP or anti-Clathrin. B. Hek293 cells were transiently transfected with HA-tagged wild-type ubiquitin, ubiquitin K48R or ubiquitin K63R. Total cell lysates (TCL) and GHR immunoprecipitates were analysed on western blot using the indicated antibodies. EV, empty vector. C. Cells were transfected as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014676#pone-0014676-g001" target="_blank">Fig. 1A</a> and then treated for 15 min with GH at 37°C as indicated. GHR was immunoprecipitated from SDS-boiled lysates, and analysed for ubiquitination. 130, mature GHR, 110, precursor GHR. At the bottom of the figure the ratios of ubiquitinated versus 130kD-GHR are given. All data in this figure are representative of three independent experiments.