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. 2001 Nov 1;15(21):2822–2836. doi: 10.1101/gad.912901

Figure 1.

Figure 1

Figure 1

Smad3- and TGF-β-induced degradation of SnoN is mediated by the ubiquitin-dependent proteasome. (A) Pulse-chase analysis of SnoN degradation by various Smad proteins. 293T cells cotransfected with HA–SnoN and the indicated Flag-tagged Smads were subjected to pulse-chase assays as described in Materials and Methods. Immunoprecipitation was carried out with anti-Flag to purify Smad-associated SnoN or with anti-HA to purify total cellular HA–SnoN. The half-lives of the two populations were similar. As a control, SnoN from singly transfected cells was isolated by immunoprecipitation with anti-HA. Abbreviations used: S3, Smad3; S3C, MH2 domain of Smad3; S3N, MH1 domain of Smad3; S2, Smad2; S2C, MH2 domain of Smad2; S2NL, MH1 and linker domains of Smad2; S4, Smad4. (B) Proteasome inhibitors block Smad3-induced degradation of SnoN. Transfected 293T cells were pretreated with DMSO (control) or various proteolytic inhibitors for 45 min followed by a pulse-chase assay as described in Materials and Methods. Inhibitors were present throughout the duration of the pulse-chase. Quantification of 35S-labeled SnoN was carried out with the Bio-Rad Molecular Imager FX system and is shown below the gels. (C) Lactacystin blocks TGF-β-induced degradation of endogenous SnoN. Hep3B cells were pretreated with DMSO (lanes 1–3) or lactacystin (lanes 4–6) for 4 h followed by stimulation with 200 pM TGF-β1 for the indicated times. Endogenous SnoN was isolated by immunoprecipitation with an anti-SnoN anti-serum and analyzed by immunoblotting with anti-SnoN. (D) Smad2 and Smad3 induce ubiquitination of SnoN. HA–Smads or Flag–SnoN were transfected either alone or together into 293T cells and isolated by immunoprecipitation with either anti-HA (lanes 1–3) or anti-Flag (lanes 4–7). Polyubiquitination of SnoN was detected by immunoblotting of the immunoprecipitates with an anti-ubiquitin antibody (top). The amounts of SnoN (middle) and Smads present in the immunoprecipitates were shown on the two lower panels. (E) The MH2 domain of Smad3 induces SnoN ubiquitination. The experiments were carried out as described in D.