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. 2017 Dec 13;8:2116. doi: 10.1038/s41467-017-02029-7

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — OTUD1 cleaves K33-poly-ubiquitin chain on SMAD7 Lysine 220. a K220 ubiquitination of SMAD7 identified by mass spectrometry. Right: Comassie staining of Flag-SMAD7 precipitations. b Sequence alignment of the PY motif and identified K220 ubiquitination site in SMAD7 orthologs of different species. c Immunoblot (IB) of total cell lysate (TCL) and immunoprecipitates derived from Flag-SMAD7 overexpressing HEK293T cells transfected with K6-, K11-, K27-, K29-, K33-, K48-, and K63-linked HA-Ub constructs and Myc-OTUD1 as indicated. d IB of TCL and immunoprecipitation derived from HEK293T cells transfected with wt, K27-, K33-, K48-linked HA-Ub, together with Flag-SMAD7-wt or K220R mutant as indicated. e Upper panel: the model of WW domain of E3-ubiquitin ligase Smurf2 binding to PY loop (residue 203-220) of SMAD7. Middle and lower panels: the model of mono-ubiquitin (PDB ID: 2XK5) conjugation to PY loop (residue 203-220) at residue Lys220. Both WW domain (green) and PY loop (red) were shown in ribbon, ubiquitin was shown in gray surface, and the key residues involved in domain interaction are drawn in stick representations. f IB of TCL and anti-SMAD7 immunoprecipitants derived from K33-linked HA-Ub expressing HEK293T cells depleted of OTUD1. g IB of TCL and Nickle-pull down precipitants derived from K33-linked His-Ub expressing HEK293T cells transfected with Flag-SMAD7-wt/ K220R and depleted of OTUD1 as indicated. h IB of input and anti-Flag immunoprecipitates derived from HEK293T cells transfected with Flag-SMAD7-wt or K220R mutant as indicated. i IB of input and anti-Flag immunoprecipitates derived from HEK293T cells transfected with Flag-SMAD7 and depleted of OTUD1 as indicated. j CAGA₁₂-Luc transcriptional response of HEK293T cells transfected with empty vector (Co.vec), SMAD7-wt or K220R at different doses as indicated and treated with TGF-β (0.5 ng/ml) overnight. k IB of biotinylated cell surface TβRI in HeLa cells stably transfected with empty vector (Co. vec), SMAD7-wt or K220R mutant expression plasmids and treated with TGF-β (5 ng/ml) for indicated time points. Quantification of the band intensities is shown in the lower panel. Band intensity was normalized to the t = 0 controls. Results are shown as means ± SD of three independent sets of experiments. l Working model of OTUD1-mediated SMAD7 deubiquitination