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. 2020 Sep 24;10:15725. doi: 10.1038/s41598-020-72791-0

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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OTUD4 regulates the ubiquitination of the TβR complex. (A) HEK293T cells were transfected with TβRI and/or FLAG-OTUD4. After 48 h cells were lysed and immunoprecipitated with anti- TβRI affinity resin. Immunoprecipitated lysates and whole cell extracts were probed with the indicated antibodies. (B) HEK293T cells were transfected with TβRII and FLAG-OTUD4. After 48 h cells were lysed and immunoprecipitated with anti- OTUD4 affinity resin. Immunoprecipitated lysates and whole cell extracts were probed with the indicated antibodies. (C) HEK293T cells were transfected with HA-ubiquitin, TβRI and either FLAG-OTUD4, or FLAG-OTUD4 DD. After 48 h cells were lysed and immunoprecipitated with anti- TβRI affinity resin. Immunoprecipitated lysates and whole cell extracts were probed with the indicated antibodies. (D) HEK293T cells were transfected with HA-ubiquitin, TβRII and either FLAG-OTUD4, or FLAG-OTUD4 DD. After 48 h cells were lysed and immunoprecipitated with anti-TβRII affinity resin. Immunoprecipitated lysates and whole cell extracts were probed with the indicated antibodies. (E) HEK293T cells were transfected with TβRI and either FLAG-OTUD4 or FLAG OTUD4 DD. Whole cell extracts were probed with indicated antibodies. β-Actin is used as the loading control. (F) HEK293T cells were transfected with TβRII and either FLAG-OTUD4 or FLAG OTUD4 DD. Cells were stimulated where indicated with TGFβ (100 pM) overnight before lysis. Whole cell extracts were probed with indicated antibodies. β-Actin is used as the loading control. (G) HEK293T cells were co-transfected with TβRI and OTUD4 shRNA hairpins B and C. After 72 h cells were lysed and whole cell extracts were probed with indicated antibodies. β-Actin is used as the loading control. (H) HEK293T cells were co-transfected with TβRII and OTUD4 shRNA hairpins B and C. After 72 h cells were lysed and whole cell extracts were probed with indicated antibodies. β-Actin is used as the loading control. (I) HEK293T cells were co-transfected with TβRI and OTUD4 shRNA hairpin C. Cells were treated with either MG132 (10 μM) or chloroquine (400 μM) or in combination overnight before lysis. Cells were subsequently lysed and whole cell extracts were probed with indicated antibodies. β-Actin is used as the loading control. Full-length blots for all panels are shown in Supplementary Information.