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 Aug 5;7:12355. doi: 10.1038/ncomms12355

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

Copyright © 2016, The Author(s)

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

(a) PTEN treatment of EG5 for MS analysis of protein modification. Sf9-expressed FLAG-EG5 was incubated with or without His-tagged PTEN before FLAG immunopreciptation. The EG5 bands were excised from coommassie-stained gel for MS analysis of potential modification alterations. (b) A peptide of EG5 from the region aa919-aa929 identified by MS showing reduced phosphorylation at Thr926 in the PTEN-treated sample. (c) Quantification of phosphorylation at Thr926 in PTEN-treated and untreated samples. (d) Reduction of EG5 phosphorylation at Thr926 by PTEN. FLAG-EG5 treated with and without PTEN as prepared as in a was subjected to FLAG immunoprecipitation followed by immunoblotting analysis of EG5 phosphorylation using a site-specific (Thr926) phospho-EG5 antibody. (e) Elevation of EG5 phosphorylation at Thr926 in Pten null cells. Pten^+/+ and Pten^−/− MEFs were analysed for EG5 phosphorylation and abundance by immunoblotting. (f) HeLa cells containing shPTEN or control shcoo2 were released from double thymidine block (DTB) for different periods of time followed by immunoblotting analysis of EG5 phospohrylation at Thr926. The same blot was probed with EG5 antibody to show EG5 expression levels. β-Actin was used as a loading control. (g) Dose-dependent reduction of EG5 phosphorylation by PTEN in vitro. Sf9-expressed His-PTEN protein was purified using a Ni-NTA agarose column. FLAG-EG5 expressed in Sf9 cells was immunoprecipitated with anti-FLAG M2 beads and incubated with increasing amounts of His-PTEN proteins, followed by western blot analysis of EG5 phosphorylation. The same blot was probed with EG5 antibody to show EG5 levels loaded in each lane. (h) Phosphatase-dependent reduction of EG5 phosphorylation by PTEN. His-PTEN proteins (including wild type and two mutants forms, C124S and G129E) were produced in Sf9 cells and purified using a Ni-NTA agarose column. FLAG-EG5 was incubated with equal amounts of His-PTEN proteins before examination of EG5 phosphorylation. Protein input of EG5 and His-PTEN (wild type and two mutants) is shown in the middle and lower panels from re-probing of the same blot with anti-EG5 antibody. (i) Reduction of EG5 phosphorylation by PTEN in phosphatase-dependent manner. Pten^−/− MEFs were transfected with vectors encoding FLAG-tagged wild-type PTEN or the phosphatase-deficient PTEN mutants PTEN^C124S and PTEN^G129E, before immunoblotting assessment of EG5 phosphorylation at Thr926. The expression of EG5, PTEN (wild type as well as two phosphatase-deficient mutants) and β-actin was then evaluated by reblotting with corresponding antibodies.