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. 2018 Dec 26;294(8):2880–2891. doi: 10.1074/jbc.RA118.003822

Figure 4.

Figure 4.

HS-152 inhibits SMURF1-mediated ubiquitination and degradation. A, HS-152 inhibits SMURF1-mediated RHOB degradation in a dose-dependent manner. HEK293T cells were transfected with FLAG-tagged RHOB (F/RHOB) and WT or C699A FLAG-tagged SMURF1 (F/SMURF1) as indicated. After overnight treatment with or without different doses of HS-152, total cell lysates were subjected to immunoblotting using indicated antibodies. The steady-state protein levels were quantified using Image Lab software (Bio-Rad) with β-actin as a loading control. Results were plotted in right panel as the levels of F/RHOB in cells co-transfected with WT F/SMURF1 and F/RHOB at each dose of HS-152 treatment relative to the level of F/RHOB in cells transfected with F/RHOB alone and without HS-152 treatment. B, HS-152 inhibits SMURF1-mediated RHOA degradation in a dose-dependent manner. HEK293T cells transfected with indicated FLAG-tagged RHOA (F/RHOA) and F/SMURF1 (WT or C699A) were treated with different doses of HS-152 and subjected to immunoblotting and then quantified and plotted as in (A). C, HS-152 inhibits SMURF1-mediated SMAD1 degradation in a dose-dependent manner. HEK293T cells transfected with indicated FLAG-tagged SMAD1 (F/SMAD1) and F/SMURF1 (WT or C699A) were treated with a different dose of HS-152 and subjected to immunoblotting and then quantified and plotted as in (A). D, HS-152 up-regulates endogenous RHOB levels through SMURF1. HEK293T cells transfected with control shRNA (sh-Con) or shRNA against SMURF1 (sh-SMURF1) and treated 4 h with or without 2 μm HS-152 and then subjected to immunoblotting assay. The lower panel presents quantitative analysis of Western blotting results (mean ± S.D. of three independent experiments). E, HS-152 inhibits SMURF1-mediated RHOB ubiquitination in vitro. FLAG-tagged RHOB (F/RHOB) and His-tagged SMURF1 (His/SMURF1) expressed and purified from bacteria were subjected to an in vitro ubiquitination assay in the absence or presence of different doses of HS-152 as indicated. The reaction products were then subjected to anti-FLAG immunoprecipitation (IP) followed by immunoblotting assay to detect ubiquitin-conjugated RHOB ((Ub)n-RHOB) using an anti-ubiquitin antibody.