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Cell Permeable Rho Inhibitor

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Introduction

This product, Cat nr CT04, consists of highly purified C3 Transferase (Cat nr CT03) covalently linked to a proprietary cell penetrating moiety via a disulfide bond.  The cell penetrating moiety allows rapid and efficient transport through the plasma membrane.  Once in the cytosol, the cell penetrating moiety is released, thereby allowing C3 Transferase to freely diffuse intracellularly and inactive RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1.

The Exoenzyme C3 Transferase from Clostridium botulinum is commonly used to selectively inactivate the GTPases RhoA, RhoB, and RhoC, both in vivo and in vitro.  C3 Transferase inhibits Rho proteins by ADP-ribosylation on asparagine 41 in the effector binding domain of the GTPase.  A major limitation of C3 Transferase for use in in vivo applications is that this protein is only slightly cell permeable.  Consequently, overnight incubations with C3 Transferase at concentrations as high as 100 µg/ml are often necessary to inactivate Rho proteins in cultured cells.  Under these conditions, the long incubation period and large amount of C3 Transferase that are required can be disruptive to basic cellular functions and economically burdensome.  Cytoskeleton, Inc. has overcome these problems by providing a cell permeable form or C3 Transferase that can efficiently inactivate cellular Rho proteins in as little as 2 h. 


CT04 has been used to inactive Rho proteins to an efficiency of 75-95% in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized cell lines (see Table 1 in the gallery for recommended conditions of use for different cells types).

Purity

The Exoenzyme C3 Transferase from Clostridium botulinum has been produced in a bacterial expression system.  The recombinant protein contains six histidine residues at its amino terminus (His tag), has a molecular weight of approximately 24 kDa, and is greater than 90% pure.  To make the purified C3 Transferase protein cell permeable, a proprietary cell penetrating moiety has been linked via a reversible disulfide bond.  Cell Permeable Rho Inhibitor is supplied as a white lyophilized powder.

Biological Activity

Cell Permeable Rho Inhibitor (Cat nr CT04) is useful for efficient inactivation of RhoA, RhoB, and RhoC in a variety of cultured cells.  The reagent inhibits Rho proteins in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized lines.  Cells treated with Cell Permeable C3 Transferase can be subjected to any one of a number of assays that indicate a decrease in Rho activity, including focal adhesion or stress fiber (Cat nr BK005) disruption assays and Rho activity assays by G-LISA™ (Cat nr BK124) or pulldown (Cat nr BK036).  See Figures 1 and 2 in the gallery for examples of stress fiber disruption and Rho inactivation demonstrating CT04 biological activity.
The concentration of Cell Permeable C3 Transferase required for efficient inactivation of Rho proteins can vary between cell types.  In addition, the length of treatment can be manipulated to yield a moderate or robust phenotype (see Figures 1 and 2 in the gallery).  For these reasons, the concentration of this reagent and the duration of treatment should be determined by the user.  Typically the effective range is between 0.5 and 2.0 µg/ml with incubations times of 2-4 h for a moderate phenotype and 4-6 h for a robust phenotype.  Recommended conditions for several cell types are detailed in Table 1 in the gallery.

Product Uses Include


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