Kits

At Cytoskeleton, we develop signal transduction kits which are focused on small G-protein research. In particular we provide kits for measuring the activated forms of Rho and Ras family members, and their intrinsic properties of GTPase and GTP/GDP exchange.

 

Activation assays come in two types, the G-LISA 96-well format and the traditional Pull-down variety. The G-LISA format is very accurate and flexible in that the 96-wells are divisible into 8-well strips making it the most economical option for a project. The Pull-down variety has been traditionally used for testing small numbers of samples or a quick look-see experiment where low accuracy is acceptable. Unfortunately due to the low accuracy of the Pull-down technique and low endogenous activation levels in some samples (may be only 10 to 20% increase in activity levels) some positive results may not be found. We recommend utilizing the GLISA assay wherever possible due to the greater confidence obtained from higher accuracy.

Apart from Activation Assays, Cytoskeleton also provides some innovative kits for studying GEFs and GAPs. In particular the BK100 kit is useful for measuring GTP or GDP exchange in Rho family members, and it can also be used to study a new GEF or a new GEF/small G-protein combination. The Rho GAP kit, Cat. # BK105, measures the release of phosphate from GTP hydrolysis by a Rho family protein which is accelerated by the GAP protein provided in the kit.

For more information click on the datasheet below.  

Related Products

 

For more information click on the Documents tab and see the datasheet, or contact Technical Support at tservice@cytoskeleton.com .

Many publications cite the use of Cytoskeleton's kits in the Materials and Methods section of papers. Usually the citation is associated with a particular result in the form of a graph or image that helps the you, the authors, present your findings. This indicates the utility of the Kits to produce publication quality data in a short timeframe thus helping improve the productivity of your efforts. Example citations for Small G-Protein assay kits are shown below.  More citations are available on individual product pages.

 

 


 

 

G-LISA RhoA Activation Assay Biochem Kit (luminescence format) (Cat. # BK121)

 

Nobe et al., 2012. Two distinct dysfunctions in diabetic mouse mesenteric artery contraction are caused by changes in the Rho A–Rho kinase signaling pathway. E. J. Pharmacol. v 683, pp 217-225.

 

Wang et al., 2012. RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response. Cardiovascular Diabetology. v 11, p 7.

 

Zuo et al., 2012. Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells. J. Cell. Physiol. v 227, pp 1399-1407.

 

Alvarez et al. (2010). Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels. British J of Pharmacology 160 ,1326-37.

 

Heckman-Stoddard et al. (2009). Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression. Breast Cancer Research 11 ,http://breast-cancer-research.com/content/11/4/R61.

 

Hammar et al. (2009). Role of the Rho-ROCK (Rho-Associated Kinase) Signaling Pathway in the Regulation of Pancreatic β-Cell Function. Endocrinology 150 ,2072-2079.

 

Chastre et al. (2009). TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB Journal 23 ,916–928.

 

Kinoshita et al., 2008. Mol Biol Cell. 19, 2289

 

Moniz et al. (2008). WNK2 modulates MEK1 activity through the Rho GTPase pathway. Cellular Signalling 20 ,1762-68.

 

Lesato et al. (2008). Tiotropium Bromide Attenuates Respiratory Syncytial Virus Replication in Epithelial Cells. Respiration 76 ,434-441.

 

Kinoshita et al. (2008). Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube Formation. Molecular Biology of the Cell 19 ,2289-2299.

 

Scott et al., 2007. J Invest Dermatol. v 127, p 668.

 

Schreibelt et al. (2007). Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling. FASEB Journal 21 ,3666-3676.

 

Tanaka et al.  (2007). Neural Expression of G Protein-coupled Receptors GPR3, GPR6, and GPR12 Up-regulates Cyclic AMP Levels and Promotes Neurite Outgrowth. J. Biol. Chem 282 ,10506-10515.

 

Higashibata et al., 2006. BMC Biochem. 7, 19

 

Zuo et al., 2006. Biochem Biophys Res Commun. 351, 361

 

Woods and Beier, 2006. J Biol Chem. 281, 13134

 


 

 

G-LISA RhoA Activation Assay Biochem Kit (colorimetric format)  (Cat. # BK124)

 

Howe and Addison, 2012. RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA. Vascular Cell. v 4, p 1.

 

Yang and Kim, 2012. The RhoA-ROCK-PTEN pathway as a molecular switch for anchorage dependent cell behavior. Biomaterials. v 33, pp 2902-2915.

 

Garrido-Gomez et al., 2012. Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F-actin regulation. FASEB J. doi: 10.1096/fj.12-204008.

 

Greco et al., 2012. Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptor. Cardiovasc Res. doi: 10.1093/cvr/cvs204.

 

Chen et al., 2012. Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR. Biochem. Pharmacol. v 83, pp 1183-1194. 

 

Zhou et al., 2012. HSV-mediated gene transfer of C3 transferase inhibits Rho to promote axonal regeneration. Exp. Neurol. http://dx.doi.org/10.1016/j.expneurol.2012.06.016.

 

McCoy et al., 2012. Protease-activated receptor 1 (PAR1) coupling to Gq/11 but not to Gi/o or G12/13 is mediated by discrete amino acids within the receptor second intracellular loop. Cellular Signalling. v 24, pp 1351-1360.

 

Ramseyer et al., 2012. Tumor Necrosis Factor α Decreases Nitric Oxide Synthase Type 3 Expression Primarily via Rho/Rho Kinase in the Thick Ascending Limb. Hypertension. v 59, pp 1145-1150.  

 

Dhaliwal et al., 2012. Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases. PLoS ONE. v 7, e35046. 

 

Jin et al. (2011). Increased SRF Transcriptional Activity is a Novel Signature of Insulin Resistance in Humans and Mice. J Clin Invest.

 

Ganguly et al. (2011). Adiponectin Increases LPL Activity via RhoA/ROCK-Mediated Actin Remodelling in Adult Rat Cardiomyocytes. Endocrinology 152 ,247.

 

Rapier et al., 2010. Cancer Cell Int. 10, 24

 

Nini L, Dagnino L. (2010). Accurate and reproducible measurements of RhoA activation in small samples of primary cells. Anal Biochem 398 ,135-7.

 

Yang et al. (2010). Fluoride induces vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aortas. Environmental Toxicology and Pharmacology 29 ,290-296.

 

Musso et al. (2010). Relevance of the mevalonate biosynthetic pathway in the regulation of bone marrow mesenchymal stromal cell-mediated effects on T cell proliferation and B cell survival. Haematologica DOI: 10.3324/haematol.2010.031633.

 

Lichtenstein et al. (2010). Secretase-Independent and RhoGTPase/PAK/ERK-Dependent Regulation of Cytoskeleton Dynamics in Astrocytes by NSAIDs and Derivatives. J Alz Dis 22 ,1135.

 

Ridgway et al. (2010). Modulation of GEF-H1 Induced Signaling by Heparanase in Brain Metastatic Melanoma Cells. J Cellular Biochemistry 111 ,1299-1309.

 

Fang et al. (2010). Allogeneic Human Mesenchymal Stem Cells Restore Epithelial Protein Permeability in Cultured Human Alveolar Type II Cells by Secretion of Angiopoietin-1. J Biol Chem 285 ,26211-26222.

 

Romero et al. (2010). Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture. Toxicol. Sci. 118 ,602-612.

 

Rapier et al. (2010). The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells. Cancer Cell International 10 ,24.


Hammar et al. (2009). Role of the Rho-ROCK (Rho-Associated Kinase) Signaling Pathway in the Regulation of Pancreatic β-Cell Function. Endocrinology 150 ,2072-2079.


Chastre et al. (2009). TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB Journal 23 ,916–928.

 

Ramirez et al., 2008. J Immunol. 180, 1854

 

Sequeira et al. (2008). Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapedesis. Clinical and Experimental Metastatis 25 ,569-579.

 

Moore et al. (2008). Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1. Development 135 ,2855-2864.

 

Kinoshita et al. (2008). Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube Formation. Molecular Biology of the Cell 19 ,2289-2299.

 

Seifert et al. (2008). Differential activation of Rac1 and RhoA in neuroblastoma cell fractions. Neurosci Lett 450 ,176-180.

 

Korobova and Svitkina  (2008). Arp2/3 Complex Is Important for Filopodia Formation, Growth Cone Motility, and Neuritogenesis in Neuronal Cells. Mol. Biol. Cell. 19 ,1561-1574.

 

Mercer and Helenius (2008). Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells. Science 320 ,531.

 

Keely et al., 2007. Methods Enzymol. v 426, p 27.

 

Scott et al., 2007. J Invest Dermatol. v 127, p 668.

 

Schreibelt et al. (2007). Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling. FASEB Journal 21 ,3666-3676.



Tanaka et al.  (2007). Neural Expression of G Protein-coupled Receptors GPR3, GPR6, and GPR12 Up-regulates Cyclic AMP Levels and Promotes Neurite Outgrowth. J. Biol. Chem 282 ,10506-10515.

 

Bradley et al., 2006. Mol Biol Cell. 17, 4827


 

G-LISA Rac 1,2,3 Activation Assay Biochem Kit (colorimetric format) (Cat. # BK125)

 

Dhaliwal et al., 2012. Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases. PLoS ONE. v 7, e35046. 

 

Halpert et al. (2011). Rac-dependent doubling of HeLa cell area and impairment of cell migration and cell cycle by compounds from Iris germanica. Protoplasma DOI: 10.1007/s00709-010-0254-1.

 

Vives et al. (2011). The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts. Journal of Bone and Mineral Research 26 ,1099.

 

Tanaka et al., 2010. Biochem Biophys Res Commun. v 399, p 677.

 

Johanna et al. (2010). Rac1 activity changes are associated with neuronal pathology and spatial memory long-term recovery after global cerebral ischemia. Neurochem International 57 ,762-773.

 

Lichtenstein et al. (2010). Secretase-Independent and RhoGTPase/PAK/ERK-Dependent Regulation of Cytoskeleton Dynamics in Astrocytes by NSAIDs and Derivatives. J Alz Dis 22 ,1135.

 

Ridgway et al. (2010). Modulation of GEF-H1 Induced Signaling by Heparanase in Brain Metastatic Melanoma Cells. J Cellular Biochemistry 111 ,1299-1309.

 

Fang et al. (2010). Allogeneic Human Mesenchymal Stem Cells Restore Epithelial Protein Permeability in Cultured Human Alveolar Type II Cells by Secretion of Angiopoietin-1. J Biol Chem 285, 26211-26222.

 

Romero et al. (2010). Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture. Toxicol. Sci. 118 ,602-612.

 

Baumer et al., 2009. J Cell Physiol. 220, 716

 

Chastre et al. (2009). TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB Journal 23 ,916–928.

 

Kawther Abu-Elneel, Tomoyo Ochiishi, Miguel Medina, Monica Remedi, Laura Gastaldi, Alfredo Caceres, and Kenneth S. Kosik (2008). A delta-Catenin Signaling Pathway Leading to dendritic protrusions. J Biol Chem 283 ,32781-32791.

 

Mercer and Helenius (2008). Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells. Science 320 ,531.

 

Pontow et al., 2007. Virology. 368, 1

 


 

 

G-LISA Rac 1 Activation Assay Biochem Kit (luminescence format)  (Cat. # BK126)

 

Binker et al., 2011. TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2. Biochem. Biophys. Res. Comm. v 405, pp 140-145.

 

Sayedyahossein et al., 2012. Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes. FASEB J. doi: 10.1096/fj.12-207852.

 

Kikuchi et al., 2012. Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma. Oncogene. doi:10.1038/onc.2012.46.

 

Eggers et al., 2012. STE20-related Kinase Adaptor Protein α (STRADα) Regulates Cell Polarity and Invasion through PAK1 Signaling in LKB1-null Cells. J. Biol. Chem. v 287, pp 18758-18768.

 

Vives et al. (2011). The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts. Journal of Bone and Mineral Research 26 ,1099.

 

McHenry et al. (2010). P190B RhoGAP has pro-tumorigenic functions during MMTV-Neu mammary tumorigenesis and metastasis. Breast Cancer Res.

 

Johanna et al. (2010). Rac1 activity changes are associated with neuronal pathology and spatial memory long-term recovery after global cerebral ischemia. Neurochem International 57 ,762-773.

 

Baumer et al., 2009. J Cell Physiol. 220, 716

 

Heckman-Stoddard et al. (2009). Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression. Breast Cancer Research 11 ,http://breast-cancer-research.com/content/11/4/R61.

 

Chastre et al. (2009). TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB Journal 23 ,916–928.

 

Ramirez et al., 2008. J Immunol. 180, 1854

 

Moniz et al. (2008). WNK2 modulates MEK1 activity through the Rho GTPase pathway. Cellular Signalling 20 ,1762-68.

 

Pontow et al., 2007. Virology. 368, 1

 


 

 

G-LISA Cdc42 Activation Assay Biochem Kit (colorimetric format)  (Cat. # BK127)

 

Chen et al., 2012. Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR. Biochem. Pharmacol. v 83, pp 1183-1194. 

 

Eggers et al., 2012. STE20-related Kinase Adaptor Protein α (STRADα) Regulates Cell Polarity and Invasion through PAK1 Signaling in LKB1-null Cells. J. Biol. Chem. v 287, pp 18758-18768.

 

Dhaliwal et al., 2012. Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases. PLoS ONE. v 7, e35046. 

 

Oliver et al., 2011. Br J Cancer. v 104, p 324.

 

McHenry et al. (2010). P190B RhoGAP has pro-tumorigenic functions during MMTV-Neu mammary tumorigenesis and metastasis. Breast Cancer Res.

 

Lichtenstein et al. (2010). Secretase-Independent and RhoGTPase/PAK/ERK-Dependent Regulation of Cytoskeleton Dynamics in Astrocytes by NSAIDs and Derivatives. J Alz Dis 22 ,1135.

 

Schlegel and Waschke (2010). Impaired cAMP and Rac 1 Signaling Contribute to TNF-α-induced Endothelial Barrier Breakdown in Microvascular Endothelium. Microcirculation 16 ,521.

 

Stankiewicz et al. (2010). GTPase activating protein function of p85 facilitates uptake and recycling of the β1 integrin. Biochemical and Biophysical Research Communications 391 ,443.

 

Romero et al. (2010). Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture. Toxicol. Sci. 118 ,602-612.

 

Heckman-Stoddard et al. (2009). Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression. Breast Cancer Research 11 ,http://breast-cancer-research.com/content/11/4/R61.

 


 

 

Rac1 G-LISA™ Activation Assay (Colorimetric Based)  (Cat. # BK128)

 

Antonov et al., 2012. Regulation of endothelial barrier function by TGF-β type I receptor ALK5: Potential role of contractile mechanisms and heat shock protein 90. J. Cell. Physiol. v 227, pp 759-771. 

 

Greco et al., 2012. Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptor. Cardiovasc Res. doi: 10.1093/cvr/cvs204.

 

Oshikawa et al., 2012. Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells. Am. J. Physiol. Heart Circ. Physiol. v 302, pp H724-H732.

 

Chen et al., 2012. Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR. Biochem. Pharmacol. v 83, pp 1183-1194. 

 

Montalvo-Ortiz et al., 2012. Characterization of EHop-016, a novel small molecule inhibitor of Rac GTPase. J. Biol. Chem. v 287, pp 13228-13238.

 

Stefanini et al., 2012. Rap1-Rac1 Circuits Potentiate Platelet Activation. Arterioscler Thromb Vasc Biol. v 32, pp 434–441.

 

Vives et al. (2011). The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts. Journal of Bone and Mineral Research 26 ,1099.

 

 

 


 

 

Ras G-LISA™ Activation Assay (Colorimetric Based)  (Cat. # BK131)

 

Gil-Henn et al., 2012. Arg/Abl2 promotes invasion and attenuates proliferation of breast cancer in vivo. Oncogene. doi:10.1038/onc.2012.284

 

 

 


 

 

 

Ras Activation Assay Biochem Kit (bead pull down format) (Cat. # BK008)

 

Stoppa et al., 2012. Ras signaling contributes to survival of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Tax-positive T-cells. Apoptosis. v 17, pp 219-228.

 

Jiang et al., 2010. Activation of Rho GTPases in Smith–Lemli–Opitz syndrome: pathophysiological and clinical implications. Hum. Mol. Gen. v 19, pp 1347–1357.

 

Kowluru and Kowluru, 2007. Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina. Mol. Vis. v 13, pp 602-10.

 

Kowluru, 2010. Role of matrix metalloproteinase-9 in the development of diabetic retinopathy and its regulation by H-Ras. Invest. Ophthalmol. Vis. Sci. v 51, pp 4320-4326. 

 

Lito et al., 2008. Evidence that sprouty 2 is necessary for sarcoma formationby H-Ras oncogene-transformed human fibroblasts. J. Biol. Chem. v 283, pp 2002-2009.

 

Rose et al., 2010. Stimulatory effects of the multi-kinase inhibitor sorafenib on human bladdercancer cells. Br. J. Pharmacol. v 160, pp 1690–1698.

 

Wang et al., 2007. Investigation of the immunosuppressive activity of artemether on T-cell activation and proliferation. Br. J. Pharmacol. v 150, pp 652–661.

 

 


 

 

Cdc42 Activation Assay Biochem Kit (bead pull down format) (Cat. # BK034)

 

Kawther Abu-Elneel, Tomoyo Ochiishi, Miguel Medina, Monica Remedi, Laura Gastaldi, Alfredo Caceres, and Kenneth S. Kosik (2008). A delta-Catenin Signaling Pathway Leading to dendritic protrusions. J Biol Chem 283 ,32781-32791.

 

Nur-E-Kamal, A., Ahmed, I., Kamal, J., Schindler, M. and Meiners, S. (2005). Three dimensional nanofibrillar surfaces induce activation of Rac. Biochem. Biophys. Res. Commun. 331, 428-434.

 

Slice, L. W., Chiu, T. and Rozengurt, E. (2005). Angiotensin II and epidermal growth factor induce cyclooxygenase-2 expression in intestinal epithelial cells through small GTPases using distinct signaling pathways. J. Biol. Chem. 280, 1582-1593.

 

Tang, D. D., Zhang, W. and Gunst, S. J. (2005). The Adapter Protein CrkII Regulates Neuronal Wiskott-Aldrich Syndrome Protein, Actin Polymerization, and Tension Development during Contractile Stimulation of Smooth Muscle. J. Biol. Chem. 280, 23380-23389.

 

Liu, X. F., Ishida, H., Raziuddin, R. and Miki, T. (2004). Nucleotide exchange factor ECT2 interacts with the polarity protein complex Par6/Par3/protein kinase Cζ (PKCζ) and regulates PKCζ activity. Mol. Cell. Biol. 24, 6665-6675.

 

Sasai, N., Nakazawa, Y., Haraguchi, T. and Sasai, Y. (2004). The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. 6, 741-748.

Tang, D. D. and Gunst, S. J. (2004). The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle. J. Biol. Chem. 279, 51722-51728.

 

 


 

 

Rac1 Activation Assay Biochem Kit  (bead pull down format) (Cat. # BK035)

 

Slice, L. W., Chiu, T. and Rozengurt, E. (2005). Angiotensin II and epidermal growth factor induce cyclooxygenase-2 expression in intestinal epithelial cells through small GTPases using distinct signaling pathways. J. Biol. Chem. 280, 1582-1593.

 

Sasai, N., Nakazawa, Y., Haraguchi, T. and Sasai, Y. (2004). The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. 6, 741-748.

 

Yang, S. A., Carpenter, C. L. and Abrams, C. S. (2004). Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in platelets. J. Biol. Chem. 279, 42331-42336.

 

Zhang, Y., Chen, K., Tu, Y. and Wu, C. (2004). Distinct roles of two structurally closely related focal adhesion proteins, α-parvins and β-parvins, in regulation of cell morphology and survival. J. Biol. Chem. 279, 41695-41705.

 

Chromy, B. A., Nowak, R. J., Lambert, M. P., Viola, K. L., Chang, L., Velasco, P. T., Jones, B. W., Fernandez, S. J., Lacor, P. N., Horowitz, P. et al. (2003). Self-assembly of Aβ(1-42) into globular neurotoxins. Biochemistry 42, 12749-12760.

 

Quadri, S. K., Bhattacharjee, M., Parthasarathi, K., Tanita, T. and Bhattacharya, J. (2003). Endothelial barrier strengthening by activation of focal adhesion kinase. J. Biol. Chem. 278, 13342-13349.

 

 

 


 

RhoA Activation Assay Biochem Kit (bead pull down format) (Cat. # BK036)

 

Khan et al. (2011). Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice. J Clin Invest doi:10.1172/JCI43758.

 

Yi H, Tao L, Feng TX, Ken C, Ming LL. (2010). Effects of ischemic preconditioning on vascular reactivity and calcium sensitivity after hemorrhagic shock and their relationship to the Rho A-Rho-kinase pathway in rats. J Cardiovasc Pharmacol.

 

Pixley, F. J., Xiong, Y., Yu, R. Y., Sahai, E. A., Stanley, E. R. and Ye, B. H. (2005). BCL6 suppresses RhoA activity to alter macrophage morphology and motility. J. Cell Sci. 118, 1873-1883.

 

Birukova, A. A., Liu, F., Garcia, J. G. and Verin, A. D. (2004). Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly. Am. J. Physiol. 287, L86-93.

 

Cetin, S., Ford, H. R., Sysko, L. R., Agarwal, C., Wang, J., Neal, M. D., Baty, C., Apodaca, G. and Hackam, D. J. (2004). Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions. J. Biol. Chem. 279, 24592-24600.

 

Orr, A. W., Pallero, M. A., Xiong, W. C. and Murphy-Ullrich, J. E. (2004). Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly. J. Biol. Chem. 279, 48983-48992.

 

Sasai, N., Nakazawa, Y., Haraguchi, T. and Sasai, Y. (2004). The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. 6, 741-748.

 

Setiadi, H. and McEver, R. P. (2003). Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow. J. Cell Biol. 163, 1385-1395.

 


 

Arf6 Activation Assay Biochem Kit (bead pull down format) (Cat. # BK033-S)

 

Berg-Larsen A., et al. 2013. Differential regulation of Rab GTPase expression in monocyte-derived dendritic cells upon lipopolysaccharide activation: A correlation to maturation-dependent functional properties. PLoS ONE. 8: e73538. 

 

 


 

 

CytoPhos™ Endpoint Phosphate Assay (Cat. # BK054)

 

Funk, C. J., Davis, A. S., Hopkins, J. A. and Middleton, K. M. (2004). Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators. Anal. Biochem. 329, 68-76.

Question 1:  I'm expecting to see only a small difference in RhoA activation.  What is the best way to measure this and what sensitivity can I expect?

Answer 1:  To detect small differences in RhoA activation between control and treated conditions, we recommend either the absorbance or luminescence-based RhoA G-LISA activation assays (Cat. # BK124 or BK121, respectively).  The absorbance-based RhoA G-LISA (Cat. # BK124) has a detection limit of 0.050 ng activated RhoA and a linear detection range of 0.05 – 2 ng activated RhoA.  The coefficient of variation (8 replicates) is 12%.  The luminescence-based RhoA G-LISA (Cat. # BK121) is recommended for use with 3-D cultures grown in matrigel or collagen matrices because of the additional sensitivity this kit offers.  The luminescence-based RhoA G-LISA has a detection limit of 0.025 ng activated RhoA and a linear range of 0.025-1 ng activated RhoA.

 

Question 2:  I have a GEF protein immunoprecipitate.  How can I measure the GEF activity in this sample?

Answer 2:  After eluting your protein of interest from the beads, the protein’s GEF activity can be measured using Cytoskeleton’s RhoGEF Exchange Assay Biochem Kit (Cat. # BK100).  Cytoskeleton Inc. has developed a mant fluorophore-based GEF assay designed for characterizing GEFs and identifying GEF inhibitors.  This kit contains human Cdc42 (Cat. # CD01), Rac1 (Cat. # RC01) and RhoA (Cat. # RH01) proteins and the GEF domain of Dbs (Cat. # GE01) as a positive control GEF for Cdc42 and RhoA.  The kit also comes with a 384-well and 96-well plate along with exchange buffer that contains the mant-GTP.  Once bound to GTPases, the fluorophore emission intensity of mant-GTP increases dramatically (approximately 2 fold). Therefore, the enhancement of fluorescent intensity in the presence of small GTPases and GEFs will reflect the respective GEF activities of known or unknown proteins.  We recommend titrating the concentration of your GEF protein to optimize its activity.  We suggest a titration range of 0.2-1 uM.  Here is a citation that used our GEF assay kit with an immunoprecipitated protein: Kakiashvili et al., 2009.  GEF-H1 mediates tumor necrosis factor-a-induced Rho activation and myosin phosphorylation: role in the regulation of tubular paracellular permeability.  J. Biol. Chem. 284, 11454-11466.

 

For more information, click on the Documents tab above and see the datasheet, or contact Technical Support at tservice@cytoskeleton.com

Page
  1. RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) - 3 x 10 assays BK030
    RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) - 3 x 10 assays
    Learn More
  2. Arf1 G-LISA Activation Assay Kit (Colorimetric Based) - 96 assays BK132
    G-LISA Arf1 Activation Assay Biochem Kit (Absorbance Based)
    Learn More
  3. Arf1 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK032-S
    Arf1 Activation Assay Biochem Kit (bead pull-down format) - 20 Assays
    Learn More
  4. Arf6 G-LISA Activation Assay Kit (Colorimetric Based) - 96 assays BK133
    G-LISA Arf6 Activation Assay Biochem Kit (Absorbance Based)
    Learn More
  5. Arf6 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK033-S
    Arf6 Activation Assay Biochem Kit (bead pull-down format) - 20 Assays
    Learn More
  6. Cdc42 G-LISA Activation Assay (Colorimetric format) - 24 assays BK127-S
    G-LISA Cdc42 Activation Assay Biochem Kit (colorimetric format)
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