Fig. 4

Fbw7 interacts with and ubiquitinates Stat3. a Fbw7 interacts with Stat3 at exogenous levels. Immunoblotting analysis of lysates after immunoprecipitation from HEK293T cells transfected with Myc-Fbw7 and Flag-Stat3. b Fbw7 interacts with Stat3 at endogenous levels. Cell lysates from SU-DHL-2 cells were immunoprecipitated with anti-Fbw7 or anti-Stat3 antibody, followed by immunoblotting with anti-Fbw7 or anti-Stat3 antibody, respectively. IgG was used as a control. c Fbw7 interacts with pStat3^Tyr705 at endogenous levels. Cell lysates from SU-DHL-2 cells were immunoprecipitated with anti-Fbw7 or anti-pStat3^Tyr705 antibody, followed by immunoblotting with anti-Fbw7 or anti- pStat3^Tyr705 antibody, respectively. d and e, Flag-Stat3 and HA-ubiquitin were co-expressed with Myc-Fbw7 in HEK293T cells. After cells were treated with or without 10 mM MG132 for 6 h, Stat3 and pStat3^Tyr705 were immunoprecipitated with anti-Flag or anti-pStat3^Tyr705 antibody, and the polyubiquitination status of Stat3 and pStat3^Tyr705 was detected by immunoblotting. f similar to E, cells were treated with 10 ng/L IL6 for 6 h to activate Stat3 signaling. The experiments were repeated three times, and representative images of blots are shown