Product Uses Include
Rac and Cdc42 Activator (Cat. # CN02) is useful for efficient activation of Rac1, Rac2, Rac3 and Cdc42 in a variety of cultured cells. The reagent activates Rac and Cdc42 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 Rac or Cdc42 activity, including membrane ruffles and lamellipodia (Rac) staining (Cat. # BK005 and Figure 2) and Rac or Cdc42 activity assays by G-LISA™ (Cat. # BK125 and BK127 resp.) See Figure 1 for example of Cdc42 activation measured by the G-LISA assay. EGF is standardized in CN02 by measurement in units, thus 100 ng of EGF is 1 unit of CN02.
Rac/Cdc42 Activator II, epidermal growth factor (EGF) acts through a tyrosine kinase receptor (EGFR) to rapidly stimulate actin reorganization at the cell membrane resulting in membrane ruffling. This primary morphological response, which occurs within 1-10 minutes in Swiss 3T3 cells, has been shown to be mediated through activation of Rac (1). Biochemical assays that quantitate the amount of active (GTP-bound) small G-proteins have demonstrated that both Rac and Cdc42 are rapidly activated (within 30 seconds–2minutes) by EGF stimulation (2, 3). A later, secondary response to EGF treatment is the formation of actin stress fibers which is mediated through the activation of Rho (1). EGF is a useful tool in studying Rho signaling pathways, it should be noted, that EGF activates several other important signal transduction pathways including Ras/Raf/MAPK, JAK/STAT and PI3K/AKT and data should be interpreted accordingly.
1) Ridley A. et al. 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70,401-410.
2) Tu S. et al. 2003. Epidermal growth factor-dependent regulation of Cdc42 is mediated by the Src tyrosine kinase. J. Biol. Chem. 278,49293-49300.
3) Kim et al. 2008. Epidermal growth factor-induced enhancement of glioblatoma cell migration in 3D arises from an intrinsic increase in speed but an extrinsic matrix- and proteolysis-dependent increase in persistence. Mol Bio Cell. 19, 4249-4259.
Rac and Cdc42 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.
At 0.25 to 0.5 unit / ml CN02 will activate Rac by 1.5 to 4 fold in epithelial, endothelial, hematopoietic and primary human cell types as measured by the G-LISATM Rac Activation Assay (Cat. BK125) and observed by ruffles and lamellipodia formation (see Figure 2B). At 0.5 to 1.0 unit / ml CN02 will activate Cdc42 by 1.25 to 2.5 fold in epithelial, endothelial, hematopoietic and primary human cell types as measured by the G-LISATM Cdc42 Activation Assay (Cat. BK127, see Figure 1).
Swiss 3T3 cells were serum starved (SS) for 16 h at 1% serum and 8 h with 0% serum and treated with CN01 (0.1, 0.5 and 1.0 units/ml for 1.5, 3.0, 6.0, 10 and 30 min). Cell lysates subjected to the Cdc42 G-LISA™ (BK127) assay and OD was read at 490 nm. The "controlled state" serum starved value (0.22) was subtracted from these samples prior to plotting. At 1.0 unit/ml the total activation was 2.05 fold or 105% over the controlled state at 1.5 min.
The concentration of CN02 activator required for efficient activation of Rac and Cdc42 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 activation (see Tables 1 and 2). 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 0.1 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 CN02 treated versus untreated samples because there are activators in the serum added to cultured cells. Inconjunction, incubation times of 1 to 10 min should be tested for each cell type. Recommended conditions for several cell types are detailed in Tables 1 and 2.
Table 1. Suggested Conditions for Rac activation in serum free medium. The indicated cells were subjected to Rac activation assay with CN02 in serum free medium. Serum containing medium will cause activation in all samples which will override effects seen by CN02 alone. Optimal conditions were determined by manipulating reagent concentration and duration of treatment.
1. A moderate phenotype is characterized by a 80-150% increase in Rac activity accompanied by ruffles and lamellipodia formation (see Figure 2b). A robust phenotype is characterized by >200% increase in Rac activity accompanied by a similar ruffles and lamellipodia formation.
2. Rac activation usually precedes the structural changes in cell morphology by 3 to 10min.
Table 2. Suggested concentrations for Cdc42 activation in serum free medium
Note: A moderate phenotype is characterized by a 25-50% increase in Cdc42 activity (see Figure 2B). A robust phenotype is characterized by >50% increase in Cdc42 activity.
Swiss 3T3 fibroblasts plated on coverslips at 1000 cells / cm2 and grown for two days in DMEM plus 10% fetal calf serum at 37°C and 5% CO2, were serum starved for 16 h in media containing 1% serum and 8 h in 0% serum media. Cultures were treated with 5 µl of CN02 per ml of medium for 10 min at 37°C. Cells were then fixed, stained with rhodamine-labeled phalloidin (Cat. # PHDR1 or BK005), and visualized by fluorescence microscopy. Images were taken at a magnification of 40×. The untreated control cells were treated with 5ul sterile PBS per ml of medium. The cells treated with CN02 produced abundant ruffles and lamellipodia whereas the control had less than 10% of CN02 levels of similar actin structures. Under similar conditions the activity of Cdc42 and Rac increased by 50 and 130% respectively as measured by the G-LISATM Activation Assays (Cat.# BK127 and BK125 resp.). A = serum starved cell, 2 s exposure; B = example Rac activation, 0.3 s exposure
Valtcheva et al., 2013. The orphan adhesion G protein coupled receptor GPR97 regulates migration of lymphatic endothelial cells via the small GTPases RhoA and Cdc42. J. Biol. Chem. doi: 10.1074/jbc.M113.512954.
Wan et al., 2013. Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts. Biochem. Biophys. Res. Commun. doi: http://dx.doi.org/10.1016/j.bbrc.2013.03.020.
Zhou et al., 2012. A hypermorphic missense mutation in PLCG2, encoding phospholipase Cγ2, causes a dominantly inherited autoinflammatory disease with immunodeficiency. Am. J. Hum. Genet. 91, 713-720.
Monteleon et al., 2012. Establishing epithelial glandular polarity: interlinked roles for ARF6, Rac1, and the matrix microenvironment. Mol. Biol. Cell. 23, 4495-4505.
Dhaliwal et al., 2012. Cellular cytoskeleton dynamics modulates non-viral gene delivery through Rho GTPases. PLoS ONE. 7(4): e35046. doi:10.1371/journal.pone.0035046.
Anderson et al. 2011. The L6 domain tetraspanin Tm4sf4 regulates endocrine pancreas differentiation and directed cell migration. Development. 138, 3213-3224.
Question 1: What is the chemical nature of the Rac/Cdc42 activator CN02?
Answer 1: CN02 is epidermal growth factor (EGF). The EGF is greater than 95% pure and supplied as a white lyophilized powder. One unit of CN02 is equivalent to 100 ng of EGF.
Question 2: Does CN02 selectively activate Rac or Cdc42?
Answer 2: CN02 (epidermal growth factor) will activate both Rac and Cdc42 indirectly in many different types of cells. In addition, EGF is involved in activating many other signal transduction cascades including MAPK, Akt and JNK. For a direct Rho/Rac/Cdc42 activator, please see Cat. # CN04.
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