Product Uses Include
This product consists of highly purified C3 Transferase (Cat. # CT03) covalently linked to a proprietary cell penetrating moiety via a disulfide bond. The cell penetrating moiety allows rapid and efficient transport through the plasma membrane. Once in the cytosol, the cell penetrating moiety is released, thereby allowing C3 Transferase to freely diffuse intracellularly and inactive RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1.
The Exoenzyme C3 Transferase from Clostridium botulinum is commonly used to selectively inactivate the GTPases RhoA, RhoB, and RhoC, both in vivo and in vitro. C3 Transferase inhibits Rho proteins by ADP-ribosylation on asparagine 41 in the effector binding domain of the GTPase. A major limitation of C3 Transferase for use in in vivo applications is that this protein is only slightly cell permeable. Consequently, overnight incubations with C3 Transferase at concentrations as high as 100 µg/ml are often necessary to inactivate Rho proteins in cultured cells. Under these conditions, the long incubation period and large amount of C3 Transferase that are required can be disruptive to basic cellular functions and economically burdensome. Cytoskeleton, Inc. has overcome these problems by providing a cell permeable form or C3 Transferase that can efficiently inactivate cellular Rho proteins in as little as 2 h.
CT04 has been used to inactive Rho proteins to an efficiency of 75-95% in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized cell lines (see Table 1 for recommended conditions of use for different cells types).
|Table 1. Suggested Conditions for Rho inactivation by Cell Permeable C3 Transferase. The indicated cells were subjected to Rho inactivation assays with CT04. Note: These concentrations were determined in serum free medium. For cells grown in serum containing medium the recommended concentration is four times higher.|
2.0 µg/ml, 2 h (1, 2)
2.0 µg/ml, 4-6 h (1, 2)
0.5 µg/ml, 2 h (1, 2)
0.5 µg/ml, 4-6 h (1, 2)
1.0 µg/ml, 2 h (1, 2)
1.0 µg/ml, 4-6 h (1, 2)
1.0 µg/ml, 2 h (1, 2)
1.0 µg/ml, 4-6 h (1, 2)
1.0 µg/ml, 2 h (2)
1.0 µg/ml, 4 h (2)
1.0 µg/ml, 4 h (2)
1.0 µg/ml, 4-6 h (1, 2)
* A moderate phenotype is characterized by a 10-40% decrease in Rho activity accompanied by stress fiber disruption and a well spread morphology (see Figures 1 and 2).
** A robust phenotype is characterized by a >50% decrease in Rho activity accompanied by a loss of stress fibers, cell body collapse, and protrusion of dendrite-like extensions (see Figures 1 and 2).
1 Based on morphological assays examining cell shape and the architecture of the actin cytoskeleton.
2 Based on biochemical data from Rho activity assays.
The Exoenzyme C3 Transferase from Clostridium botulinum has been produced in a bacterial expression system. The recombinant protein contains six histidine residues at its amino terminus (His tag), has a molecular weight of approximately 24 kDa, and is greater than 90% pure (see Cat. # CT03 for gel purity). To make the purified C3 Transferase protein cell permeable, a proprietary cell penetrating moiety has been linked via a reversible disulfide bond. Cell Permeable Rho Inhibitor is supplied as a white lyophilized powder.
Cell Permeable Rho Inhibitor (Cat. # CT04) is useful for efficient inactivation of RhoA, RhoB, and RhoC in a variety of cultured cells. The reagent inhibits Rho proteins in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized lines. Cells treated with Cell Permeable C3 Transferase can be subjected to any one of a number of assays that indicate a decrease in Rho activity, including focal adhesion or stress fiber (Cat. # BK005) disruption assays and Rho activity assays by G-LISA™ (Cat. # BK124) or pulldown (Cat. # BK036). See Figures 1 and 2 for examples of stress fiber disruption and Rho inactivation demonstrating CT04 biological activity.
Figure 1. Cell permeable Rho inhibitor disrupts stress fibers and can be manipulated to induce either moderate or robust phenotypes. Swiss 3T3 fibroblasts plated on coverslips were untreated (A) or treated with 2.0 µg/ml of CT04 for 2 h (B) or 4 h (C) at 37°C. Cell were then fixed, stained with Rhodamine-labeled Phalloidin (Cat. # BK005), and visualized by flu orescence microscopy. Images were taken at a magnification of 20×. The untreated control cells in A were well spread and stress fibers were present. The cells treated for 2 h in B displayed a Moderate Phenotype, characterized by a loss of stress fibers, cells remaining well spread, and a 10-40% decrease in Rho activity (also see Figure 2). Treatment for 4 h (C) yielded a Robust Phenotype, characterized by a loss of stress fibers, decreased cell spreading, collapse of the cell body, protrusion of dentritic extensions, and a >50% decrease in Rho activity (also see Figure 2).
Figure 2a. CT04 inhibition of Rho activity as measured with the RhoA G-LISA Activation Assay (Cat.# BK124). Serum starved Swiss 3T3 fibroblasts were untreated (no CT04) or trea ted with 0.20, 0.50 and 2.0 µg/ml of CT04 for 4h in serum free medium at 37°C, then activated with 100µg/ml calpeptin for 10min. Cells were then lysed and RhoA activity was measured by the RhoA G-LISA Activation Assay (Cat.# BK124). Note: At 2.0 µg/ml CT04 for 4h results in almost complete (90%) inhibition of RhoA activity.
Figure 2b. Cell permeable Rho Inhibitor decreases RhoA activity. Swiss 3T3 fibroblasts were untreated (no CT04) or treated with 2.0 µg/ml of CT04 for 2 h (CT04, 2 h) or 4 h (CT04, 4 h) at 37°C. Cells were then lysed and RhoA activity was measured by pulldown assay using the Rho-binding domain of the Rho effector Rhotekin (RhoA Activation Assay Biochem Kit, Cat. # BK036). The pulldowns (active RhoA) and cell extracts (total RhoA) were analyzed by SDS-PAGE followed by Western blotting with a RhoA specific antibody. The level of RhoA activity in each sample is proportional to the amount of RhoA precipitated in the pulldowns (active RhoA, upper panel).
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|Vadakumchery, Anila et al.||The Small GTPase RHOA Links SLP65 Activation to PTEN Function in Pre B Cells and Is Essential for the Generation and Survival of Normal and Malignant B Cells||Frontiers in Immunology||2022||ISSN 1664-3224|
|Kuromiya, Keisuke et al.||Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells||Cell Reports||2022||ISSN 2211--1247|
|Hauke, Michael et al.||Active RhoA Exerts an Inhibitory Effect on the Homeostasis and Angiogenic Capacity of Human Endothelial Cells||Journal of the American Heart Association||2022||ISSN 2047-9980|
|Sakabe, Masahide et al.||Inhibition of β1-AR/Gαs signaling promotes cardiomyocyte proliferation in juvenile mice through activation of RhoAYAP axis||eLife||2022|
|Caballero, D. et al.||Quantifying protrusions as tumor-specific biophysical predictors of cancer invasion in in vitro tumor micro-spheroid models||In vitro models 2022 1:3||2022||ISSN 2731--3441|
|Filina, Yuliya et al.||MAP kinases in regulation of NOX activity stimulated through two types of formyl peptide receptors in murine bone marrow granulocytes||Cellular Signalling||2022|
|Xue, Tan et al.||Effects of Aster B-mediated intracellular accumulation of cholesterol on inflammatory process and myocardial cells in acute myocardial infarction||Hellenic Journal of Cardiology||2022|
|Skerrett-Byrne, David A. et al.||Global profiling of the proteomic changes associated with the post-testicular maturation of mouse spermatozoa||Cell Reports||2022||ISSN 2211--1247|
|Lachowski, Dariusz et al.||Substrate Stiffness-Driven Membrane Tension Modulates Vesicular Trafficking via Caveolin-1||ACS Nano||2022||ISSN 1936-086X|
|Wong, Darren Chen Pei et al.||BNIP-2 Activation of Cellular Contractility Inactivates YAP for H9c2 Cardiomyoblast Differentiation||Advanced Science||2022||ISSN 2198--3844|
|Leguay, Kévin et al.||Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry||Journal of Cell Biology||2022||ISSN 1540-8140|
|Caire, Robin et al.||YAP promotes cell-autonomous immune responses to tackle intracellular Staphylococcus aureus in vitro||Nature Communications 2022 13:1||2022||ISSN 2041--1723|
|Sharif, Muhammad et al.||Porcine Sapovirus-Induced Tight Junction Dissociation via Activation of the RhoA/ROCK/MLC Signaling Pathway||Journal of Virology||2021||ISSN 0022--538X|
|Palander, Oliva et al.||Nonredundant roles of DIAPHs in primary ciliogenesis||Journal of Biological Chemistry||2021||ISSN 1083-351X|
|Bian, Rui et al.||Research paper rac gtpase activating protein 1 promotes gallbladder cancer via binding DNA ligase 3 to reduce apoptosis||International Journal of Biological Sciences||2021||ISSN 1449-2288|
|Tan, Dandan et al.||RhoA-GTPase Modulates Neurite Outgrowth by Regulating the Expression of Spastin and p60-Katanin||Cells||2020||ISSN 2073-4409|
|Che, Pulin et al.||Neuronal Wiskott-Aldrich syndrome protein regulates Pseudomonas aeruginosa-induced lung vascular permeability through the modulation of actin cytoskeletal dynamics||FASEB journal : official publication of the Federation of American Societies for Experimental Biology||2020||ISSN 1530--6860|
|Yong, Yu et al.||Regulation of degenerative spheroids after injury||Scientific Reports||2020||ISSN 2045-2322|
|Palander, Oliva et al.||DIAPH1 regulates ciliogenesis and trafficking in primary cilia||FASEB Journal||2020||ISSN 1530-6860|
|Kolyvushko, Oleksandr et al.||Equine alphaherpesviruses require activation of the small gtpases rac1 and cdc42 for intracellular transport||Microorganisms||2020||ISSN 2076-2607|
|Laplagne, Chloé et al.||Vγ9Vδ2 T Cells Activation Through Phosphoantigens Can Be Impaired by a RHOB Rerouting in Lung Cancer||Frontiers in Immunology||2020||ISSN 1664-3224|
|Salgado-Lucio, Monica L. et al.||FAK regulates actin polymerization during sperm capacitation via the ERK2/GEF-H1/RhoA signaling pathway||Journal of Cell Science||2020||ISSN 1477-9137|
|Rom, Slava et al.||Author Correction: Hyperglycemia and advanced glycation end products disrupt BBB and promote occludin and claudin-5 protein secretion on extracellular microvesicles (Scientific Reports, (2020), 10, 1, (7274), 10.1038/s41598-020-64349-x)||Scientific Reports||2020||ISSN 2045-2322|
|Xia, Mingyu et al.||Activation of the RhoA-YAP-β-catenin signaling axis promotes the expansion of inner ear progenitor cells in 3D culture||Stem Cells||2020||ISSN 1549-4918|
|Ramírez-Ramírez, Danelia et al.||Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa||Journal of cellular biochemistry||2020||ISSN 1097--4644|
|Lundin, Vanessa et al.||YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biomechanical Forces of Blood Flow||Developmental Cell||2020||ISSN 1878-1551|
|Kim, Sarah Hyun Ji et al.||Integrin crosstalk allows CD4+ T lymphocytes to continue migrating in the upstream direction after flow||Integrative biology : quantitative biosciences from nano to macro||2019||ISSN 1757-9708|
|Tocci, Piera et al.||β-arrestin1/YAP/mutant p53 complexes orchestrate the endothelin A receptor signaling in high-grade serous ovarian cancer||Nature Communications||2019||ISSN 2041-1723|
|Kalappurakkal, Joseph Mathew et al.||Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading||Cell||2019||ISSN 1097-4172|
|Eliazer, Susan et al.||Wnt4 from the Niche Controls the Mechano-Properties and Quiescent State of Muscle Stem Cells||Cell Stem Cell||2019||ISSN 1875-9777|
|Li, Xu et al.||A positive feedback loop of profilin-1 and RhoA/ROCK1 promotes endothelial dysfunction and oxidative stress||Oxidative Medicine and Cellular Longevity||2018||ISSN 1942-0994|
|Wang, Jinyang et al.||Rho A Regulates Epidermal Growth Factor-Induced Human Osteosarcoma MG63 Cell Migration||International journal of molecular sciences||2018||ISSN 1422--0067|
|Han, Han et al.||Regulation of the Hippo Pathway by Phosphatidic Acid-Mediated Lipid-Protein Interaction||Molecular Cell||2018||ISSN 1097-4164|
|Soliman, Mahmoud et al.||Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells||Scientific Reports||2018||ISSN 2045-2322|
|Herbert, Lindsay M. et al.||RhoA increases ASIC1a plasma membrane localization and calcium influx in pulmonary arterial smooth muscle cells following chronic hypoxia||American Journal of Physiology - Cell Physiology||2018||ISSN 1522-1563|
|Marikawa, Y. et al.||RHOA activity in expanding blastocysts is essential to regulate HIPPO-YAP signaling and to maintain the trophectoderm-specific gene expression program in a ROCK/actin filament-independent manner||Molecular Human Reproduction||2018||ISSN 1460-2407|
|Sánchez-Martín, David et al.||Effects of DLC1 deficiency on endothelial cell contact growth inhibition and angiosarcoma progression||Journal of the National Cancer Institute||2018||ISSN 1460-2105|
|Freeman, Spencer A. et al.||Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement||Cell||2018||ISSN 1097-4172|
|Platet, Nadine et al.||The tumor suppressor CDX2 opposes pro-metastatic biomechanical modifications of colon cancer cells through organization of the actin cytoskeleton||Cancer Letters||2017||ISSN 1872-7980|
|Yu, Lixia et al.||C-Maf inducing protein inhibits coflin-1 activity and alters podocyte cytoskeleton organization||Molecular Medicine Reports||2017||ISSN 1791-3004|
|Durgan, Joanne et al.||Mitosis can drive cell cannibalism through entosis||eLife||2017||ISSN 2050-084X|
|Wu, Mengrui et al.||Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signalling pathway||Nature Communications||2017||ISSN 2041-1723|
|Tod, Jo et al.||Pro-migratory and TGF-β-activating functions of αvβ6 integrin in pancreatic cancer are differentially regulated via an Eps8-dependent GTPase switch||Journal of Pathology||2017||ISSN 1096-9896|
|Prieto, Catalina P. et al.||Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton's jelly mesenchymal stem cells (WJ-MSC)||Stem Cell Research and Therapy||2017||ISSN 1757-6512|
|Lázaro-Diéguez, Francisco et al.||Cell-cell adhesion accounts for the different orientation of columnar and hepatocytic cell divisions||Journal of Cell Biology||2017||ISSN 1540-8140|
|Hendrick, Janina et al.||The polarity protein Scribble positions DLC3 at adherens junctions to regulate Rho signaling||Journal of Cell Science||2016||ISSN 1477-9137|
|Rom, Slava et al.||PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrier||Journal of Neuroinflammation||2016||ISSN 1742-2094|
|Alarcon, Vernadeth B. et al.||Statins inhibit blastocyst formation by preventing geranylgeranylation||Molecular Human Reproduction||2016||ISSN 1460-2407|
|Wagener, Brant M. et al.||Neuronal Wiskott-Aldrich syndrome protein regulates TGF-β1-mediated lung vascular permeability||FASEB Journal||2016||ISSN 1530-6860|
|Lu, Xuanyu et al.||Ameloblastin, an Extracellular Matrix Protein, Affects Long Bone Growth and Mineralization||Journal of Bone and Mineral Research||2016||ISSN 1523-4681|
|Slauson, Sarah R. et al.||Viral vector effects on exoenzyme C3 transferase-mediated actin disruption and on outflow facility||Investigative Ophthalmology and Visual Science||2015||ISSN 1552-5783|
|Rom, Slava et al.||Poly(ADP-ribose) polymerase-1 inhibition in brain endothelium protects the blood-brain barrier under physiologic and neuroinflammatory conditions||Journal of Cerebral Blood Flow and Metabolism||2015||ISSN 1559-7016|
|Tang, Xiao et al.||HCLOCK Causes Rho-Kinase-Mediated Endothelial Dysfunction and NF-κ B-Mediated Inflammatory Responses||Oxidative Medicine and Cellular Longevity||2015||ISSN 1942-0994|
|Su, Chien Chia et al.||Phenotypes of trypsin- and collagenase-prepared bovine corneal endothelial cells in the presence of a selective rho kinase inhibitor, Y-27632||Molecular Vision||2015||ISSN 1090-0535|
|Li, Shufeng et al.||Microtopographical features generated by photopolymerization recruit RhoA/ROCK through TRPV1 to direct cell and neurite growth||Biomaterials||2015||ISSN 1878-5905|
|Aifuwa, Ivie et al.||Senescent stromal cells induce cancer cell migration via inhibition of RhoA/ROCK/myosin-based cell contractility||Oncotarget||2015||ISSN 1949-2553|
|Manukyan, Arkadi et al.||A complex of p190RhoGAP-A and anillin modulates RhoA-GTP and the cytokinetic furrow in human cells||Journal of Cell Science||2015||ISSN 1477-9137|
|Carey, Shawn P. et al.||Comparative mechanisms of cancer cell migration through 3D matrix and physiological microtracks||American Journal of Physiology - Cell Physiology||2015||ISSN 1522-1563|
|Rom, Slava et al.||The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 ltr inhibition and diminution in Rho GTPase activity||Frontiers in Microbiology||2015||ISSN 1664-302X|
|Luo, Jixian et al.||8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and α-smooth muscle actin polymerization||Free Radical Biology and Medicine||2014||ISSN 1873-4596|
|Chen, Xiaofei et al.||The TMEFF2 tumor suppressor modulates integrin expression, RhoA activation and migration of prostate cancer cells||Biochimica et Biophysica Acta - Molecular Cell Research||2014||ISSN 1879-2596|
|Rachner, Tilman D. et al.||Dickkopf-1 is regulated by the mevalonate pathway in breast cancer||Breast Cancer Research||2014||ISSN 1465-5411|
|Wilhelm, Imola et al.||Role of Rho/ROCK signaling in the interaction of melanoma cells with the blood-brain barrier||Pigment Cell and Melanoma Research||2014||ISSN 1755-1471|
|Herr, Michael J. et al.||Tetraspanin CD9 regulates cell contraction and actin arrangement via RhoA in human vascular smooth muscle cells||PLoS ONE||2014||ISSN 1932-6203|
|DiScipio, Richard G. et al.||Complement C3a signaling mediates production of angiogenic factors in mesenchymal stem cells||Journal of Biomedical Science and Engineering||2013||ISSN 1937--6871|
|Rom, Slava et al.||Selective activation of cannabinoid receptor 2 in leukocytes suppresses their engagement of the brain endothelium and protects the blood-brain barrier||American Journal of Pathology||2013||ISSN 0002-9440|
|Brusés, Juan L.||Cell Surface Localization of α3β4 Nicotinic Acetylcholine Receptors Is Regulated by N-Cadherin Homotypic Binding and Actomyosin Contractility||PLoS ONE||2013||ISSN 1932-6203|
|Lin, Yi et al.||Angiopoietin-like 3 induces podocyte f-actin rearrangement through integrin α v β 3 /FAK/PI3K pathway-mediated rac1 Activation||BioMed Research International||2013||ISSN 2314-6133|
|Sakai, Norihiko et al.||LPA 1-induced cytoskeleton reorganization drives fibrosis through CTGF-dependent fibroblast proliferation||The FASEB Journal • Research Communication FASEB J||2013||Article Link|
|Kalia, Manjula et al.||Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic Mechanism||Journal of Virology||2013||ISSN 0022--538X|
|Huang, Bo et al.||MiRNA-125a-3p is a negative regulator of the RhoA-actomyosin pathway in A549 cells||International Journal of Oncology||2013||ISSN 1019-6439|
|Gadepalli, Ravisekhar et al.||Novel role for p21-activated kinase 2 in thrombin-induced monocyte migration||Journal of Biological Chemistry||2013||ISSN 0021-9258|
|Yugawa, Takashi et al.||Noncanonical NOTCH Signaling Limits Self-Renewal of Human Epithelial and Induced Pluripotent Stem Cells through ROCK Activation||Molecular and Cellular Biology||2013||ISSN 0270--7306|
|Shoval, Irit et al.||Antagonistic activities of Rho and Rac GTPases underlie the transition from neural crest delamination to migration||Developmental Dynamics||2012||ISSN 1058-8388|
|Ponsaerts, Raf et al.||RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system||PLoS ONE||2012||ISSN 1932-6203|
|Kshitiz et al.||Matrix rigidity controls endothelial differentiation and morphogenesis of cardiac precursors||Science Signaling||2012||ISSN 1945-0877|
|Ginestier, Christophe et al.||Mevalonate metabolism regulates basal breast cancer stem cells and is a potential therapeutic target||Stem Cells||2012||ISSN 1066-5099|
|Kim, Min Jung et al.||Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells||Biochemical and biophysical research communications||2012||ISSN 1090--2104|
|Zhu, Ying Ting et al.||Nuclear p120 catenin unlocks mitotic block of contactinhibited human corneal endothelial monolayers without disrupting adherent junctions||Journal of Cell Science||2012||ISSN 0021-9533|
|Horowitz, Jeffrey C. et al.||Survivin expression induced by endothelin-1 promotes myofibroblast resistance to apoptosis||International Journal of Biochemistry and Cell Biology||2012||ISSN 1357-2725|
|Chin, Elizabeth et al.||Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial Factors||PLoS ONE||2012||ISSN 1932-6203|
|Zhang, Weiwei et al.||Self-assembling peptide nanofiber scaffold enhanced with RhoA inhibitor CT04 improves axonal regrowth in the transected spinal cord||Journal of Nanomaterials||2012||ISSN 1687-4110|
|Wan, Qingwen et al.||Regulation of myosin activation during cell-cell contact formation by Par3-Lgl antagonism: Entosis without matrix detachment||Molecular Biology of the Cell||2012||ISSN 1059-1524|
|Balzer, Eric M. et al.||Physical confinement alters tumor cell adhesion and migration phenotypes||FASEB Journal||2012||ISSN 1530-6860|
|Steward, Robert L. et al.||Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblasts||Scientific Reports||2011||ISSN 2045-2322|
|Fan, Huapeng et al.||Macrophage Migration Inhibitory Factor and CD74 Regulate Macrophage Chemotactic Responses via MAPK and Rho GTPase||The Journal of Immunology||2011||ISSN 0022--1767|
|Navarro, Angels et al.||Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42||American Journal of Physiology - Lung Cellular and Molecular Physiology||2011||ISSN 1040-0605|
|Anderson, Keith R. et al.||The L6 domain tetraspanin Tm4sf4 regulates endocrine pancreas differentiation and directed cell migration||Development||2011||ISSN 0950-1991|
|Kakudo, Natsuko et al.||Effect of C3 transferase on human adipose-derived stem cells||Human cell||2011||ISSN 1749--0774|
|Boettcher, Jan Peter et al.||Tyrosine-phosphorylated caveolin-1 blocks bacterial uptake by inducing Vav2-RhoA-mediated cytoskeletal rearrangements||PLoS Biology||2010||ISSN 1544-9173|
|Pattabiraman, Padmanabhan P. et al.||Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells||American Journal of Physiology - Cell Physiology||2010||ISSN 0363-6143|
|Martinelli, Roberta et al.||ICAM-1-mediated endothelial nitric oxide synthase activation via calcium and AMP-activated protein kinase is required for transendothelial lymphocyte migration||Molecular Biology of the Cell||2009||ISSN 1059-1524|
|Bunda, Severa et al.||Aldosterone stimulates elastogenesis in cardiac fibroblasts via mineralocorticoid receptor-independent action involving the consecutive activation of Gα13, c-Src, the insulin-like growth factor-I receptor, and phosphatidylinositol 3-kinase/Akt||Journal of Biological Chemistry||2009||ISSN 0021-9258|
|Stirling, Lee et al.||Dual roles for RhoA/Rho-kinase in the regulated trafficking of a voltage-sensitive potassium channel||Molecular Biology of the Cell||2009||ISSN 1059-1524|
|Kim, Woo Yang et al.||Statins decrease dendritic arborization in rat sympathetic neurons by blocking RhoA activation||Journal of Neurochemistry||2009||ISSN 0022-3042|
|Kanada, Masamitsu et al.||Novel functions of Ect2 in polar lamellipodia formation and polarity maintenance during "contractile ring-independent" cytokinesis in adherent cells||Molecular Biology of the Cell||2008||ISSN 1059-1524|
|Navarro, Angels et al.||T1α/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cells||American Journal of Physiology - Lung Cellular and Molecular Physiology||2008||ISSN 1040-0605|
|Groysman, Maya et al.||A negative modulatory role for rho and rho-associated kinase signaling in delamination of neural crest cells||Neural Development||2008||ISSN 1749-8104|
|Kirchner, Marieluise et al.||Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes||Molecular Microbiology||2008||ISSN 0950-382X|
|Rupp, Paul A. et al.||A role for RhoA in the two-phase migratory pattern of post-otic neural crest cells||Developmental Biology||2007||ISSN 0012-1606|
Question 1: Can the Rho inhibitor CT04 be used with cells growing in culture?
Answer 1: Yes, Cytoskeleton modified the exoenzyme C3 Transferase from Clostridium botulinum to be cell permeable (Cat. # CT04) for the specific purpose of inhibiting Rho in living cells in as little as 2 hours.
The exoenzyme C3 Transferase from Clostridium botulinum is commonly used to selectively inactivate the GTPases RhoA, RhoB, and RhoC, both in vivo and in vitro. C3 Transferase inhibits Rho proteins by ADP-ribosylation on asparagine 41 in the effector binding domain of the GTPase. A major limitation of the standard C3 Transferase for use in in vivo applications is that this protein is not cell permeable.
Cytoskeleton’s cell permeable Rho inhibitor consists of highly purified C3 Transferase covalently linked to a proprietary cell penetrating moiety via a disulfide bond. The cell penetrating moiety allows rapid and efficient transport through the plasma membrane. Once in the cytosol, the cell penetrating moiety is released, thereby allowing C3 Transferase to freely diffuse intracellularly and inactive RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1.
Question 2: How can I assess whether Rho activity is changing in my cells following CT04 treatment?
Answer 2: There are multiple ways to measure changes in Rho activity. To visualize a change in Rho activity, we recommend examining Rho-mediated stress fiber formation with fluorescently-labeled phalloidin (Cat. # PHDG1, PHDH1, PHDN1, PHDR1). These Acti-stain phalloidins label F-actin stress fibers. Activation of Rho can be directly quantified with one of our activation assays, either the traditional pull-down (Cat. # BK036) or the RhoA G-LISA activation assay (Cat. # BK124).
If you have any questions concerning this product, please contact our Technical Service department at firstname.lastname@example.org.