Rho Activator (Cat nr CN01) is useful for efficient activation of RhoA, RhoB, and RhoC in a variety of cultured cells. The reagent activates Rho proteins in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized lines. Cells treated with the activator can be subjected to any one of a number of assays that indicate an increase in Rho activity, including focal adhesion or stress fiber staining (Cat nr BK005) and Rho activity assays by G-LISA™ (Cat nr BK124). See Figure 1 in the gallery for example of Rho activation measured by the G-LISA assay.
There are many activators of RhoA, B and C proteins in mammalian cells. Commonly used ones are calf serum, lysophospatidic acid (LPA) and calpeptin. Through years of experience in Rho activation assay, Cytoskeleton Inc. has identified calpeptin as a compound that activates many cell types and has a long timespan of activation for ease of use so this is the active component in Rho Activator Cat nr CN01. Note: Calpeptin is used as an inhibitor of calpain, but it also inhibits myosin light chain phosphorylation which is connected to stress fiber formation and hence possibly to RhoA activation.
Rho Activator is greater than 95% pure which is supplied as a white lyophilized powder.
The lyophilized protein can be stored at 4°C or -70°C with less than 10% humidity for 6 months.
The effects of CN01 can be mitigated by the use of a Rho Inhibitor such as Cat nr CT04, a reagent that efficiently inactivates cellular Rho proteins in as little as 2 h. Unit Definition: Using CN01 at 1 unit / ml will activate Rho by 1.5 to 3 fold in epithelial, endothelial, hematopoietic and primary human cell types as measured by the G-LISATM Rho Activation Assay (see Figure 1 in the gallery) and observed by stress fiber formation (see Figure 2 in the gallery).
The concentration of Rho Activator required for efficient activation of Rho proteins can vary between cell types and whether the medium contains serum or not. In addition, the length of treatment can be manipulated to yield a moderate or robust phenotype (see Table 1 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 0.25 units / ml and 1.0 unit/ml for incubation in serum free medium. In media containing serum it might be difficult to observe the difference between CN01 treated versus untreated samples because there are activators in the serum added to cultured cells. Inconjunction, incubation times of 5 to 10 min create a moderate phenotype and 10 to 20 min create a robust phenotype. Recommended conditions for several cell types are detailed in Table 1 in the gallery.
Study the effects of Rho activation on the rearrangement of the actin cytoskeleton.
Investigate the effects of Rho activation with respect to cross talk to other signal transduction pathways.