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. Author manuscript; available in PMC: 2018 Mar 15.
Published in final edited form as: Cancer Res. 2017 Jan 23;77(6):1383–1394. doi: 10.1158/0008-5472.CAN-16-2012

Figure 5.

Figure 5

USP9x selective binding to SMAD4 in competition with TIF1γ facilitated nuclear SMAD4 retention, formation of the nuclear SMAD3-SMAD4 complex and target gene expression. A, MCF-7 cells stably expressing control shRNA or USP9x shRNA were incubated with BSA or PA in the presence or absence of TGF-β1 for 2 h. Nuclear SMAD4, total USP9x and Twist protein expression were analyzed by immunoblotting. B, Nuclear SMAD4-SMAD3 and SMAD4-TIF1γ interaction were determined by immunoprecipitation with SMAD4 antibody, followed by immunoblotting with SMAD3 or TIF1γ antibody. C, MCF-7 cells were transfected with WT-SMAD4, HA-ubiquitin, and the indicated shRNAs. The cells were treated with PA in the presence or absence of TGF-β1 for 2 h and nuclear extracts were made. The SMAD4 monoubiquitination (Mono-Ub) was detected by anti-SMAD4 immunoprecipitation and immunoblot with HA-ubiquitin. *p<0.01 vs shControl/-TGF-β/-PA; #p<0.01 vs shControl/−PA; $p<0.01 vs shControl/+PA.