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04 Nov
Rac and Cdc42 Activator

 

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. 

There are many activators of Rac and Cdc42 proteins in mammalian cells. Commonly used ones are lysophosphatidylcholine (Rac, ref.1), bradykinin (Cdc42, ref.2), epidermal growth factor (EGF, Rac and Cdc42, ref.3) and tumor necrosis factor (TNF, Cdc42, ref.3). Through years of experience in Rac and Cdc42 Activation Assays, Cytoskeleton Inc. has identified EGF as a compound that activates many cell types and has a robust signal compared to basal levels. EGF is standardized in CN02 by measurement in units, thus 100 ng of EGF is 1 unit of CN02.

 

Product Uses Include

    1. Positive control for Rac and Cdc42 activation studies.

    2. Study the effects of Rac and Cdc42 activation on cell motility

    3. Study the effects of Rac activation on the rearrangement of the actin cytoskeleton.

    4. Investigate the effects of Rac and Cdc42 activation with respect to cross talk to other signal transduction pathways.



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).

 























Figure 1. Time course of Cdc42 activation with CN02.  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.

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

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.



Figure 2. CN02 Activator induces ruffles and lamellipodia in Swiss 3T3 cells.  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.

References

1) Scott G. Arioka M. and Jacobs S. 2007. Lysophosphatidylcholine mediates melanocyte dendricity through PKC zeta activation. Jo. Invest. Dermat. 127, 668-675.

2) Kozma R, Ahmed S, Best A. and Lim L. 1995. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol. Cell. Biol. 15(4), 1942-1952.

3) Ridley, AJ. & Hall, A. 1992. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389-399.

 

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