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. 2010 Sep 15;21(18):3232–3246. doi: 10.1091/mbc.E09-05-0408

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© 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).

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Figure 3. — Three essential residues for GAP domain in inducing cell rounding. (A) HeLa cells were transfected with HA-tagged PGAP or the various mutants indicated, fixed after 20 h, and subjected to confocal fluorescence microscopy with anti-HA and Alexa Fluor 488 dye–conjugated goat anti-rabbit IgG as described in Materials and Methods. (B) To determine the Rho GTPase activity, HeLa cells were transfected with FLAG-tagged RhoA alone or with various HA-tagged PGAP or its various mutants. Cell were lysed and incubated with GST fusion of the Rho-binding domain of rhotekin immobilized on beads as described in Materials and Methods. Bound active RhoA were resolved on SDS-PAGE and detected by immunoblotting with FLAG-antibody (top panel). Equal loading of GST fusion proteins is shown in the second panel.