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

. 2016 Mar 29;2:16001. doi: 10.1038/celldisc.2016.1

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

Copyright © 2016 SIBS, CAS

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

PMC Copyright notice

USP9x regulates AMOT ubiquitylation. (a) Immunoblots of HEK293T cells transfected to express HA-AMOT and V5-tagged USP9x. Cell lysates were immunoprecipitated using anti-HA to pull down HA-p130-AMOT or with anti-V5 to pull down V5 AMOT. Blots were probed with anti-V5 to detect USP9x-V5 (input) and with anti USP9x (IP). Note that the V5 antibody pulls down predominantly the shorter form of USP9x. Both long and short forms of USP9x were recovered by IP with HA-AMOT. (b) Immunoblots of HEK293T cell lysates immunoprecipitated using anti-AMOT or anti-USP9x or with non-immune IgG as a control. Blots were probed with anti-AMOT and with anti-USP9x. The recovery of endogenous AMOT by IP was very efficient. However, recovery of UPS9x by IP was very weak, which limits the sensitivity of detection of AMOT by USP9x IP. (c) Immunoblots of HEK293T cells transfected to express HA-AMOT with Myc-tagged Ubiquitin, and shRNA vectors to deplete USP9x or control shRNAs. Cells were treated with MG132 to block proteasome activity. Lysates were immunoprecipitated with anti-HA to recover AMOT and blots were probed to detect Ubiquitin (anti-Myc), AMOT (anti-HA) or with anti-Actin to control for loading. Anti-USP9x was used to monitor the efficiency of shRNA-mediated depletion in the total cell lysate. Depletion of USP9x was efficient. Note that the total level of ubiquitylated protein in the cell was unchanged in the total cell lysates after USP9x depletion. IP, immunoprecipitation.