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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 2. — The GAP domain of p50RhoGAP induces cell rounding by inactivating Rho GTPases (A) HeLa cells were treated with Rho inhibitor C3 Transferase followed by rhodamine-conjugated phalloidin staining and confocal fluorescence microscopy analysis. (B) Cells were transfected with FLAG-p50RhoGAP in the presence or absence of HA-tagged expression constructs of Cdc42, Rac1, and RhoA. Lysates were immunoprecipitated (IP) with anti-FLAG beads, and the associated proteins were separated on SDS-PAGE, blotted, and probed with HA antibody. Expression of FLAG-p50RhoGAP and HA-tagged Cdc42, Rac1, and RhoA were verified by Western blot analyses for the whole cell lysates (WCL) using anti-FLAG (third panel) and anti-HA (bottom panel), respectively. The bound GTPase was detected by anti-HA (top panel), and equal loading of IP beads were verified by anti-FLAG (second panel). (C) To determine the Rho GTPase activity, HeLa cells were transfected with FLAG-tagged wild-type RhoA in the presence and absence of HA-tagged p50RhoGAP, NBCH, or PGAP 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. (D) HeLa cells were transfected with plasmids encoding HA-RhoA alone or with FLAG-tagged full-length p50RhoGAP, PGAP, or NBCH mutants. Cells were then fixed after 20 h and subjected to confocal fluorescence microscopy as described in Materials and Methods The actin filaments were detected by direct costaining with rhodamine-conjugated phalloidin. (E) HeLa cells were cotransfected with HA-tagged PGAP mutant and wild-type or constitutively active RhoA-G14V. Cells were then fixed and images analyzed by confocal fluorescence microscopy after direct staining with rhodamine-conjugated phalloidin for actin filaments.

Figure 2. — The GAP domain of p50RhoGAP induces cell rounding by inactivating Rho GTPases (A) HeLa cells were treated with Rho inhibitor C3 Transferase followed by rhodamine-conjugated phalloidin staining and confocal fluorescence microscopy analysis. (B) Cells were transfected with FLAG-p50RhoGAP in the presence or absence of HA-tagged expression constructs of Cdc42, Rac1, and RhoA. Lysates were immunoprecipitated (IP) with anti-FLAG beads, and the associated proteins were separated on SDS-PAGE, blotted, and probed with HA antibody. Expression of FLAG-p50RhoGAP and HA-tagged Cdc42, Rac1, and RhoA were verified by Western blot analyses for the whole cell lysates (WCL) using anti-FLAG (third panel) and anti-HA (bottom panel), respectively. The bound GTPase was detected by anti-HA (top panel), and equal loading of IP beads were verified by anti-FLAG (second panel). (C) To determine the Rho GTPase activity, HeLa cells were transfected with FLAG-tagged wild-type RhoA in the presence and absence of HA-tagged p50RhoGAP, NBCH, or PGAP 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. (D) HeLa cells were transfected with plasmids encoding HA-RhoA alone or with FLAG-tagged full-length p50RhoGAP, PGAP, or NBCH mutants. Cells were then fixed after 20 h and subjected to confocal fluorescence microscopy as described in Materials and Methods The actin filaments were detected by direct costaining with rhodamine-conjugated phalloidin. (E) HeLa cells were cotransfected with HA-tagged PGAP mutant and wild-type or constitutively active RhoA-G14V. Cells were then fixed and images analyzed by confocal fluorescence microscopy after direct staining with rhodamine-conjugated phalloidin for actin filaments.