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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. 4 — OTUD1 interacts with and deubiquitinates SMAD7 in vitro. a Effect of OTUD1-wt and CA mutant (left panel) or sh-OTUD1 #1 (right panel) on CAGA₁₂-Luc transcriptional response induced by TGF-β (2.5 ng/ml) for 16 h in HEK293T cells. Co., control sgRNA. b Left panel: BLI of three representative mice injected with MDA-MB-231 control cells or cells deficient in SMAD7 (sg-SMAD7) that ectopically expressed with control vector (Co.vec) or OTUD1, images were taken 6 weeks after injection. Middle panel: BLI signals of all mice in each experimental group at week 6. Right panel: the percentage of metastasis-free mice in each experimental group followed in time. c Purified SMAD7 and OTUD1-wt/CA interaction in vitro. Prokaryotic purified SMAD7 and eukaryotic purified OTUD1 proteins were incubated and immunoprecipitated with anti-OTUD1 (left panel) or with anti-SMAD7 (right panel) antibodies. Immunoprecipitates were then immunoblotted for OTUD1 and SMAD7. d IB analysis of total cell lysate (TCL) and immunoprecipitates derived from HEK293T cells transfected with Myc-OTUD1 and Flag-SMAD1-7 constructs as indicated. e IB of anti-SMAD7 immunoprecipitate derived from HEK293T cells. Association between OTUD1 and SMAD7 was examined. 5% total cell lysates was loaded as input. f–h OTUD1 deubiquitinates poly-ubiquitination of SMAD7 in vitro. Flag-SMAD7 and HA-Ub were transfected in HEK293T cells. Poly-ubiquitinated SMAD7 was then immunoprecipitated with anti-Flag M2 beads and incubated with purified OTUD1-wt/CA protein as indicated time. Lysates were analyzed by IB with anti-HA-Ub, anti-SMAD7, and anti-OTUD1 antibodies f. Flag-SMAD7 and His-Ub were transfected into HEK293T cells. Subsequently, poly-ubiquitinated SMAD7 from the cell lysate was pulled down by Nickle beads and incubated with purified OTUD1-wt/CA for 60 mins. Lysates were analyzed by IB with anti-SMAD7 and anti-OTUD1 antibodies, asterisk indicates free SMAD7 g. His-SMAD7 was first incubated with E1, E2 (UbcH6), GST-RNF12, and ubiquitin in vitro for 3 h at 37 °C. The poly-ubiquitinated His-SMAD7 was immunoprecipitated by anti-SMAD7 antibody then incubated with purified OTUD1-wt/CA for 60 mins. The assay mixtures were analyzed by anti-Ub, anti-SMAD7 and anti-OTUD1 antibodies (h). *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed Student′s t-test a, b or two-way analysis of variance (ANOVA) b)