Product Uses Include
Rho Activator I(Cat. # 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. # BK005) and Rho activity assays by G-LISA™ (Cat. # BK124). See Figure 1 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 (1), lysophosphatidic acid (LPA)(1) and calpeptin (2). 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 I Cat.# 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.
Aguilar et al., 2011. Phos-Tag-Based Analysis of Myosin Regulatory Light Chain Phosphorylation in Human Uterine Myocytes. PLoS ONE. 6, e20903.
Question 1: What is the chemical nature of the Rho activator CN01?
Answer 1: CN01 is calpeptin and has the peptide sequence of Z-Leu-Nle-CHO. The molecular weight is 362.5 and the CAS number is 117591-20-5. Calpeptin is supplied as a lyophilized white solid and each vial contains 1 mg (10 units) of CN01. The purity is ≥95% as determined by HPLC.
Question 2: Does CN01 selectively activate Rho?
Answer 2: No, CN01 (calpeptin) does not specifically activate Rho. Calpeptin-induced Rho activation is indirect via a mechanism involving inhibition of Shp-2 phosphatase. Inhibition of Shp-2 allows constitutive activation of Rho GEFs. CN01 also inhibits calpain-1 in a mechanism that is unrelated to Rho activation. For a direct and specific Rho activator, please see Cat. # CN03.
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