Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2023 Jul 19;13:11670. doi: 10.1038/s41598-023-38726-1

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2023

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/.

PMC Copyright notice

USP4 removes K48-linked polyubiquitination from TRAF6. (A) Western blot analysis of HEK293T cells transfected with Flag-TRAF6, HA-USP4, and HA-Ub for ubiquitination levels; GAPDH was used as a loading control. (B,C) HEK293T cell lysates transfected with Flag-TRAF6, HA-Ub, together with either wild-type HA-USP4 (WT) or mutated HA-USP4 (CA) were collected and immunoprecipitated with anti-Flag agarose beads. The eluted immunocomplexes was then subjected to SDS-PAGE analysis with anti-ubiquitin antibody. (D) Lysates from HEK293T cells transiently expressing Flag-tagged TRAF6 and HA-tagged ubiquitin, together with either HA-tagged USP4 or its truncated constructs (N or C) were immunoprecipitated with anti-Flag agarose beads; the eluted protein complexes was subjected to western blot analysis with anti-ubiquitin antibody. (E) HEK293T cells transfected with Flag-TRAF6, Myc-USP4, together with either WT HA-Ub or its mutants (K48 or K63) were harvested and subjected to immunoprecipitation with anti-Flag agarose beads followed by SDS-PAGE analysis with HA antibody. (F) Lysates from HEK293T cells transfected with HA-USP4 or a control vector followed by treatment with poly (I:C) (HMW; 30 μg/ml) for 8 h were subjected to immunoprecipitation with an anti-TRAF6 antibody. This was followed by western blot analysis of eluted immunocomplexes with K48 linkage-specific ubiquitin antibodies. (G) HEK293T cells transfected with Flag-TRAF6, control siRNA or USP4-specific siRNA, together with either WT HA-Ub or its mutants (K48 or K63) were harvested and subjected to immunoprecipitation with anti-Flag agarose beads followed by SDS-PAGE analysis with anti-ubiquitin antibody. (H) Western blot analysis of RD cells transfected with control plasmid or HA-USP4 plasmid for 48 h, followed by treatment with EV71 (MOI = 0.5) for 12 h. The cell lysates were subjected to immunoprecipitation with anti-TRAF6 antibody, followed by western blot analysis of eluted immunocomplexes with K48 linkage-specific ubiquitin antibodies.