Rho activator II

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Question 1: Can the direct Rho activator CN03 be used with cells growing in culture?

Answer 1: Yes, CN03 is specifically designed to be used as a Rho activator with cultured cells. The active site of CN03 is based on the catalytic domain of the bacterial cytotoxic necrotizing factor (CNF) toxins. The catalytic domain of CN03 is covalently attached to a proprietary cell penetrating moiety. Upon entry into the cell, CN03 activates Rho GTPase isoforms by deamidating glutamine-63, which is located in the Switch II region of these GTPases. This modification converts glutamine-63 to glutamate, which blocks intrinsic and GAP-stimulated GTPase activity, resulting in constitutively active Rho. CN03 robustly increases the level of GTP-bound RhoA within 2-4 h after addition to the culture medium.

Question 2: How can I assess whether Rho activity is changing in my cells following CN03 treatment?

Answer 2: There are multiple ways to measure changes in Rho activity. To visualize a change in Rho activity, we recommend examining Rho-mediated stress fiber formation with fluorescently-labeled phalloidin (Cat. #PHDG1, PHDH1, PHDN1, PHDR1). These Acti-stain phalloidins label F-actin stress fibers. Activation of Rho can be directly quantified with one of our activation assays, either the traditional pull-down (Cat. #BK036) or the RhoA G-LISA activation assay (Cat. #BK124).

Activator for Rho pathway
Study the effects of Rho activation on cell motility
Study the effects of Rho activation on the rearrangement of the actin cytoskeleton
Investigate the effects of Rho activation with respect to cross talk with other signal transduction pathways
The G-switch Direct Modulators have been developed with an emphasis on creating highly potent cell permeable reagents that directly target the Rho family of small G-proteins. Our Direct Activator reagents are based on the catalytic domain of the bacterial CNF toxins, which are covalently attached to a proprietary cell penetrating moiety. Rho Activator II (Cat# CN03) enters the cell and activates Rho GTPase isoforms by deamidating glutamine-63, which is located in the Switch II region of these GTPases (1). This modification converts glutamine-63 to glutamate, which blocks intrinsic and GAP stimulated GTPase activity resulting in constitutively active Rho (2). Rho Activator II robustly increases the level of GTP bound RhoA within 2-4 h after addition to the culture medium, providing a more rapid alternative to transfection based methods for introducing activators like GEFs into cells. Moreover, the targeted action of Rho Activator II makes it far more attractive tool for the study of Rho GTPase signaling than classic indirect activators (e.g. LPA) that concomitantly activate other signaling pathways (e.g. Ras, PI3K and PLC).