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

. 2010 Dec 15;21(24):4312–4324. doi: 10.1091/mbc.E10-04-0310

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

© 2010 by The American Society for Cell Biology

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

PMC Copyright notice

Figure 2. — The conservation of RhoS in the structure and enzymatic activity as a Rho GTPase. (A) A schematic structural model of RhoS. Numbers indicate corresponding amino acids in sequence. (B) GTP hydrolysis activity assay. Enzymatic activity was measured by GTP hydrolysis using purified GTPase protein. The phosphate generated by hydrolysis of GTP was measured by the addition of CytoPhos (Cytoskeleton) reagent and reading the absorbance at 650 nm. Results are presented as the mean ± SD from three experiments. (C) Activated Rho GTPase (GTP-bound) protein pulldown assay. Extracts were prepared from HEK293T cells transiently expressing Flag-tagged wild-type or mutated RhoS including RhoS(D13T), RhoS(G14V), RhoS(T19N), RhoS(Q63L), and RhoS(C190S). After a 1-h incubation with GST-RBD glutathione beads, the bound proteins were analyzed by Western blotting using anti-Flag antibody. (D) A conserved C-terminal site (C190) is essential for the proper localization of RhoS. Subcellular distributions of wild-type and C190S mutant were analyzed by immunofluorescence. Twenty-four hours after transfection, MCF-7 cells transfected with the indicated plasmids were prepared for immunofluorescence analysis using anti-Flag antibody. Bar, 18.73 μm.