Rac/Cdc42 Activator II

Rac/Cdc42 Activator II
$0.00

Product Uses Include

  • Positive control for Rac and Cdc42 activation studies.
  • Study the effects of Rac and Cdc42 activation on cell motility.
  • Study the effects of Rac activation on the rearrangement of the actin cytoskeleton. 
  • Investigate the effects of Rac and Cdc42 activation with respect to cross talk to other signal transduction pathways.

Background Information

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.

References

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.

 

Material
Rac and Cdc42 Activator is greater than 95% pure which is supplied as a white lyophilized powder.

Storage
The lyophilized protein can be stored at 4°C or -70°C with less than 10% humidity for 6 months.

Biological Activity

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).

G-Switch-Logo-_White-BG_
CN02_time_course

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.

Rac_table

Notes:

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

Cdc_table

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.

CN02cellimagesmcopy

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

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Fine, Noah et al.Go with the flow: GEF-H1 mediated shear stress mechanotransduction in neutrophilsSmall GTPases2020ISSN 2154-1256
Lundin, Vanessa et al.YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biomechanical Forces of Blood FlowDevelopmental Cell2020ISSN 1878-1551
López-Posadas, Rocío et al.Inhibiting PGGT1B Disrupts Function of RHOA, Resulting in T-cell Expression of Integrin α4β7 and Development of Colitis in MiceGastroenterology2019ISSN 1528-0012
Lin, Hsiao Han et al.Lysosomal cysteine protease cathepsin S is involved in cancer cell motility by regulating store-operated Ca2+ entryBiochimica et Biophysica Acta - Molecular Cell Research2019ISSN 1879-2596
Wang, Shanshan et al.Caveolin-1 phosphorylation is essential for axonal growth of human neurons derived from iPSCsFrontiers in Cellular Neuroscience2019ISSN 1662-5102
Griesi-Oliveira, Karina et al.Actin cytoskeleton dynamics in stem cells from autistic individualsScientific Reports2018ISSN 2045-2322
Mammoto, Tadanori et al.Mesenchymal condensation-dependent accumulation of collagen VI stabilizes organ-specific cell fates during embryonic tooth formationDevelopmental Dynamics2015ISSN 1097-0177
Dubash, Adi D. et al.The GEF Bcr activates RhoA/MAL signaling to promote keratinocyte differentiation via desmoglein-1Journal of Cell Biology2013ISSN 0021-9525
Valtcheva, Nadejda et al.The orphan adhesion G protein-coupled receptor GPR97 regulates migration of lymphatic endothelial cells via the small GTPases RhoA and Cdc42Journal of Biological Chemistry2013ISSN 0021-9258
Dhaliwal, Anandika et al.Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases2012PMID 22509380
Zhou, Qing et al.A hypermorphic missense mutation in PLCG2, encoding phospholipase Cγ2, causes a dominantly inherited autoinflammatory disease with immunodeficiencyAmerican journal of human genetics2012ISSN 1537--6605
Monteleon, Christine L. et al.Establishing epithelial glandular polarity: Interlinked roles for ARF6, Rac1, and the matrix microenvironmentMolecular Biology of the Cell2012ISSN 1059-1524
Anderson, Keith R. et al.The L6 domain tetraspanin Tm4sf4 regulates endocrine pancreas differentiation and directed cell migrationDevelopment2011ISSN 0950-1991
Mammoto, Tadanori et al.Mechanochemical Control of Mesenchymal Condensation and Embryonic Tooth Organ FormationDevelopmental Cell2011ISSN 1534-5807
McAuley, Erin M. et al.Phenylboronic acid is a more potent inhibitor than boric acid of key signaling networks involved in cancer cell migrationCell Adhesion and Migration2011ISSN 1933-6926

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.

 

 If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.