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).
If you have any questions concerning this product, please contact our Technical Service department at infohoelzel.de.