Rho Inhibitor I

Rho Inhibitor I
$0.00

Product Uses Include

  • Inhibit Rho activity within 4h (compared to 24h with siRNA)
  • Stop Rho pathway signalling
  • Determine "activator" specificity for Rho pathway
  • Positive control for Rho Inhibition in cell morphology studies
  • Positive control for Rho inhibition in cell extract biochemical studies
  • Inhibit stress fiber formation
  • Inhibit RhoA, RhoB and RhoC in living cells
  • Inhibits Rho in all cell types currently tested

Material
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.

Cell Type
Cell Line
Moderate phenotype*
Robust phenotype**
Fibroblast
Swiss 3T3
2.0 µg/ml, 2 h (1, 2)
2.0 µg/ml, 4-6 h (1, 2)
Epithelial
HeLa
0.5 µg/ml, 2 h (1, 2)
0.5 µg/ml, 4-6 h (1, 2)
Epithelial
MDCK
1.0 µg/ml, 2 h (1, 2)
1.0 µg/ml, 4-6 h (1, 2)
Endothelial
HUVEC
1.0 µg/ml, 2 h (1, 2)
1.0 µg/ml, 4-6 h (1, 2)
Hematopoietic
Jurkat
1.0 µg/ml, 2 h (2)
1.0 µg/ml, 4 h (2)
Neuronal
Mouse cortex
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.

Purity
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.

Biological Activity
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.

ct04fig1

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).

CT04_results

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.

ct04fig2

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).

The concentration of Cell Permeable C3 Transferase required for efficient inactivation of Rho proteins can vary between cell types.  In addition, the length of treatment can be manipulated to yield a moderate or robust phenotype (see Figures 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 0.5 and 2.0 µg/ml with incubations times of 2-4 h for a moderate phenotype and 4-6 h for a robust phenotype.  Recommended conditions for several cell types are detailed in Table 1.

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
Sharif, Muhammad et al.Porcine Sapovirus-Induced Tight Junction Dissociation via Activation of the RhoA/ROCK/MLC Signaling PathwayJournal of Virology2021ISSN 0022--538X
Palander, Oliva et al.Nonredundant roles of DIAPHs in primary ciliogenesisJournal of Biological Chemistry2021ISSN 1083-351X
Talwar, Shefali et al.Mechanosensitive smooth muscle cell phenotypic plasticity emerging from a null state and the balance between Rac and RhoCell Reports2021ISSN 2211-1247
Bian, Rui et al.Research paper rac gtpase activating protein 1 promotes gallbladder cancer via binding DNA ligase 3 to reduce apoptosisInternational Journal of Biological Sciences2021ISSN 1449-2288
Lundin, Vanessa et al.YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biomechanical Forces of Blood FlowDevelopmental Cell2020ISSN 1878-1551
Palander, Oliva et al.DIAPH1 regulates ciliogenesis and trafficking in primary ciliaFASEB Journal2020ISSN 1530-6860
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 Reports2020ISSN 2045-2322
Laplagne, Chloé et al.Vγ9Vδ2 T Cells Activation Through Phosphoantigens Can Be Impaired by a RHOB Rerouting in Lung CancerFrontiers in Immunology2020ISSN 1664-3224
Kolyvushko, Oleksandr et al.Equine alphaherpesviruses require activation of the small gtpases rac1 and cdc42 for intracellular transportMicroorganisms2020ISSN 2076-2607
Xia, Mingyu et al.Activation of the RhoA-YAP-β-catenin signaling axis promotes the expansion of inner ear progenitor cells in 3D cultureStem Cells2020ISSN 1549-4918
Tan, Dandan et al.RhoA-GTPase Modulates Neurite Outgrowth by Regulating the Expression of Spastin and p60-KataninCells2020ISSN 2073-4409
Yong, Yu et al.Regulation of degenerative spheroids after injuryScientific Reports2020ISSN 2045-2322
Ramírez-Ramírez, Danelia et al.Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoaJournal of cellular biochemistry2020ISSN 1097--4644
Salgado-Lucio, Monica L. et al.FAK regulates actin polymerization during sperm capacitation via the ERK2/GEF-H1/RhoA signaling pathwayJournal of Cell Science2020ISSN 1477-9137
Fox, Megan E. et al.Dendritic remodeling of D1 neurons by RhoA/Rho-kinase mediates depression-like behaviorMolecular Psychiatry2020ISSN 1476-5578
Che, Pulin et al.Neuronal Wiskott-Aldrich syndrome protein regulates Pseudomonas aeruginosa-induced lung vascular permeability through the modulation of actin cytoskeletal dynamicsFASEB journal : official publication of the Federation of American Societies for Experimental Biology2020ISSN 1530--6860
Eliazer, Susan et al.Wnt4 from the Niche Controls the Mechano-Properties and Quiescent State of Muscle Stem CellsCell Stem Cell2019ISSN 1875-9777
Kim, Sarah Hyun Ji et al.Integrin crosstalk allows CD4+ T lymphocytes to continue migrating in the upstream direction after flowIntegrative biology : quantitative biosciences from nano to macro2019ISSN 1757-9708
Kalappurakkal, Joseph Mathew et al.Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell SpreadingCell2019ISSN 1097-4172
Tocci, Piera et al.β-arrestin1/YAP/mutant p53 complexes orchestrate the endothelin A receptor signaling in high-grade serous ovarian cancerNature Communications2019ISSN 2041-1723
Han, Han et al.Regulation of the Hippo Pathway by Phosphatidic Acid-Mediated Lipid-Protein InteractionMolecular Cell2018ISSN 1097-4164
Vite, Alexia et al.Α-Catenin-Dependent Cytoskeletal Tension Controls Yap Activity in the HeartDevelopment (Cambridge)2018ISSN 1477-9129
Wang, Jinyang et al.Rho A Regulates Epidermal Growth Factor-Induced Human Osteosarcoma MG63 Cell MigrationInternational journal of molecular sciences2018ISSN 1422--0067
Freeman, Spencer A. et al.Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor EngagementCell2018ISSN 1097-4172
Soliman, Mahmoud et al.Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK CellsScientific Reports2018ISSN 2045-2322
Sánchez-Martín, David et al.Effects of DLC1 deficiency on endothelial cell contact growth inhibition and angiosarcoma progressionJournal of the National Cancer Institute2018ISSN 1460-2105
Herbert, Lindsay M. et al.RhoA increases ASIC1a plasma membrane localization and calcium influx in pulmonary arterial smooth muscle cells following chronic hypoxiaAmerican Journal of Physiology - Cell Physiology2018ISSN 1522-1563
Li, Xu et al.A positive feedback loop of profilin-1 and RhoA/ROCK1 promotes endothelial dysfunction and oxidative stressOxidative Medicine and Cellular Longevity2018ISSN 1942-0994
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 mannerMolecular Human Reproduction2018ISSN 1460-2407
Durgan, Joanne et al.Mitosis can drive cell cannibalism through entosiseLife2017ISSN 2050-084X
Wu, Mengrui et al.Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signalling pathwayNature Communications2017ISSN 2041-1723
Lázaro-Diéguez, Francisco et al.Cell-cell adhesion accounts for the different orientation of columnar and hepatocytic cell divisionsJournal of Cell Biology2017ISSN 1540-8140
Itou, Junji et al.The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cellsBiochimica et Biophysica Acta - Molecular Cell Research2017ISSN 1879-2596
Yu, Lixia et al.C-Maf inducing protein inhibits coflin-1 activity and alters podocyte cytoskeleton organizationMolecular Medicine Reports2017ISSN 1791-3004
Platet, Nadine et al.The tumor suppressor CDX2 opposes pro-metastatic biomechanical modifications of colon cancer cells through organization of the actin cytoskeletonCancer Letters2017ISSN 1872-7980
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 switchJournal of Pathology2017ISSN 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 Therapy2017ISSN 1757-6512
Kempf, Anissa et al.Control of Cell Shape, Neurite Outgrowth, and Migration by a Nogo-A/HSPG InteractionDevelopmental Cell2017ISSN 1878-1551
Otsu, Keishi et al.The Semaphorin 4D-RhoA-Akt Signal Cascade Regulates Enamel Matrix Secretion in Coordination With Cell Polarization During Ameloblast DifferentiationJournal of Bone and Mineral Research2016ISSN 1523-4681
Alarcon, Vernadeth B. et al.Statins inhibit blastocyst formation by preventing geranylgeranylationMolecular Human Reproduction2016ISSN 1460-2407
Hendrick, Janina et al.The polarity protein Scribble positions DLC3 at adherens junctions to regulate Rho signalingJournal of Cell Science2016ISSN 1477-9137
Yuan, Xue et al.Ciliary IFT80 balances canonical versus non-canonical hedgehog signalling for osteoblast differentiationNature Communications2016ISSN 2041-1723
Rom, Slava et al.PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrierJournal of Neuroinflammation2016ISSN 1742-2094
Sayyad, Wasim A. et al.The role of Rac1 in the growth cone dynamics and force generation of DRG neuronsPLoS ONE2016ISSN 1932-6203
Wagener, Brant M. et al.Neuronal Wiskott-Aldrich syndrome protein regulates TGF-β1-mediated lung vascular permeabilityFASEB Journal2016ISSN 1530-6860
Lu, Xuanyu et al.Ameloblastin, an Extracellular Matrix Protein, Affects Long Bone Growth and MineralizationJournal of Bone and Mineral Research2016ISSN 1523-4681
Carey, Shawn P. et al.Comparative mechanisms of cancer cell migration through 3D matrix and physiological microtracksAmerican Journal of Physiology - Cell Physiology2015ISSN 1522-1563
Aifuwa, Ivie et al.Senescent stromal cells induce cancer cell migration via inhibition of RhoA/ROCK/myosin-based cell contractilityOncotarget2015ISSN 1949-2553
Manukyan, Arkadi et al.A complex of p190RhoGAP-A and anillin modulates RhoA-GTP and the cytokinetic furrow in human cellsJournal of Cell Science2015ISSN 1477-9137
Tang, Xiao et al.HCLOCK Causes Rho-Kinase-Mediated Endothelial Dysfunction and NF-κ B-Mediated Inflammatory ResponsesOxidative Medicine and Cellular Longevity2015ISSN 1942-0994
Li, Shufeng et al.Microtopographical features generated by photopolymerization recruit RhoA/ROCK through TRPV1 to direct cell and neurite growthBiomaterials2015ISSN 1878-5905
Slauson, Sarah R. et al.Viral vector effects on exoenzyme C3 transferase-mediated actin disruption and on outflow facilityInvestigative Ophthalmology and Visual Science2015ISSN 1552-5783
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 activityFrontiers in Microbiology2015ISSN 1664-302X
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-27632Molecular Vision2015ISSN 1090-0535
Rom, Slava et al.Poly(ADP-ribose) polymerase-1 inhibition in brain endothelium protects the blood-brain barrier under physiologic and neuroinflammatory conditionsJournal of Cerebral Blood Flow and Metabolism2015ISSN 1559-7016
Chen, Xiaofei et al.The TMEFF2 tumor suppressor modulates integrin expression, RhoA activation and migration of prostate cancer cellsBiochimica et Biophysica Acta - Molecular Cell Research2014ISSN 1879-2596
Wilhelm, Imola et al.Role of Rho/ROCK signaling in the interaction of melanoma cells with the blood-brain barrierPigment Cell and Melanoma Research2014ISSN 1755-1471
Luo, Jixian et al.8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and α-smooth muscle actin polymerizationFree Radical Biology and Medicine2014ISSN 1873-4596
Rachner, Tilman D. et al.Dickkopf-1 is regulated by the mevalonate pathway in breast cancerBreast Cancer Research2014ISSN 1465-5411
Filina, Julia V. et al.RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytesCellular Signalling2014ISSN 1873-3913
Tiftik, R. Nalan et al.Insan eritrositlerinde Rho/Rho-Kinaz yolaǧi{dotless}ni{dotless}n fonksiyonel önemiTurkish Journal of Hematology2014ISSN 1308-5263
Herr, Michael J. et al.Tetraspanin CD9 regulates cell contraction and actin arrangement via RhoA in human vascular smooth muscle cellsPLoS ONE2014ISSN 1932-6203
Rom, Slava et al.Selective activation of cannabinoid receptor 2 in leukocytes suppresses their engagement of the brain endothelium and protects the blood-brain barrierAmerican Journal of Pathology2013ISSN 0002-9440
Yugawa, Takashi et al.Noncanonical NOTCH Signaling Limits Self-Renewal of Human Epithelial and Induced Pluripotent Stem Cells through ROCK ActivationMolecular and Cellular Biology2013ISSN 0270--7306
Huang, Bo et al.MiRNA-125a-3p is a negative regulator of the RhoA-actomyosin pathway in A549 cellsInternational Journal of Oncology2013ISSN 1019-6439
Kalia, Manjula et al.Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic MechanismJournal of Virology2013ISSN 0022--538X
Lin, Yi et al.Angiopoietin-like 3 induces podocyte f-actin rearrangement through integrin α v β 3 /FAK/PI3K pathway-mediated rac1 ActivationBioMed Research International2013ISSN 2314-6133
Gadepalli, Ravisekhar et al.Novel role for p21-activated kinase 2 in thrombin-induced monocyte migrationJournal of Biological Chemistry2013ISSN 0021-9258
Hernandez-Hernandez, Victor et al.Bardet-biedl syndrome proteins control the cilia length through regulation of actin polymerizationHuman Molecular Genetics2013ISSN 0964-6906
DiScipio, Richard G. et al.Complement C3a signaling mediates production of angiogenic factors in mesenchymal stem cellsJournal of Biomedical Science and Engineering2013ISSN 1937--6871
Sakai, Norihiko et al.LPA 1-induced cytoskeleton reorganization drives fibrosis through CTGF-dependent fibroblast proliferationThe FASEB Journal • Research Communication FASEB J2013Article Link
Brusés, Juan L.Cell Surface Localization of α3β4 Nicotinic Acetylcholine Receptors Is Regulated by N-Cadherin Homotypic Binding and Actomyosin ContractilityPLoS ONE2013ISSN 1932-6203
Shoval, Irit et al.Antagonistic activities of Rho and Rac GTPases underlie the transition from neural crest delamination to migrationDevelopmental Dynamics2012ISSN 1058-8388
Kim, Min Jung et al.Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cellsBiochemical and biophysical research communications2012ISSN 1090--2104
Kshitiz et al.Matrix rigidity controls endothelial differentiation and morphogenesis of cardiac precursorsScience Signaling2012ISSN 1945-0877
Zhang, Weiwei et al.Self-assembling peptide nanofiber scaffold enhanced with RhoA inhibitor CT04 improves axonal regrowth in the transected spinal cordJournal of Nanomaterials2012ISSN 1687-4110
Ponsaerts, Raf et al.RhoA GTPase switch controls Cx43-hemichannel activity through the contractile systemPLoS ONE2012ISSN 1932-6203
Chin, Elizabeth et al.Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial FactorsPLoS ONE2012ISSN 1932-6203
Wan, Qingwen et al.Regulation of myosin activation during cell-cell contact formation by Par3-Lgl antagonism: Entosis without matrix detachmentMolecular Biology of the Cell2012ISSN 1059-1524
Ginestier, Christophe et al.Mevalonate metabolism regulates basal breast cancer stem cells and is a potential therapeutic targetStem Cells2012ISSN 1066-5099
Horowitz, Jeffrey C. et al.Survivin expression induced by endothelin-1 promotes myofibroblast resistance to apoptosisInternational Journal of Biochemistry and Cell Biology2012ISSN 1357-2725
Zhu, Ying Ting et al.Nuclear p120 catenin unlocks mitotic block of contactinhibited human corneal endothelial monolayers without disrupting adherent junctionsJournal of Cell Science2012ISSN 0021-9533
Balzer, Eric M. et al.Physical confinement alters tumor cell adhesion and migration phenotypesFASEB Journal2012ISSN 1530-6860
Navarro, Angels et al.Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42American Journal of Physiology - Lung Cellular and Molecular Physiology2011ISSN 1040-0605
Anderson, Keith R. et al.The L6 domain tetraspanin Tm4sf4 regulates endocrine pancreas differentiation and directed cell migrationDevelopment2011ISSN 0950-1991
Winzeler, Alissa M. et al.The lipid sulfatide is a novel myelin-associated inhibitor of CNS axon outgrowthJournal of Neuroscience2011ISSN 0270-6474
Steward, Robert L. et al.Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblastsScientific Reports2011ISSN 2045-2322
Kakudo, Natsuko et al.Effect of C3 transferase on human adipose-derived stem cellsHuman cell2011ISSN 1749--0774
Fan, Huapeng et al.Macrophage Migration Inhibitory Factor and CD74 Regulate Macrophage Chemotactic Responses via MAPK and Rho GTPaseThe Journal of Immunology2011ISSN 0022--1767
Pattabiraman, Padmanabhan P. et al.Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cellsAmerican Journal of Physiology - Cell Physiology2010ISSN 0363-6143
Boettcher, Jan Peter et al.Tyrosine-phosphorylated caveolin-1 blocks bacterial uptake by inducing Vav2-RhoA-mediated cytoskeletal rearrangementsPLoS Biology2010ISSN 1544-9173
Kim, Woo Yang et al.Statins decrease dendritic arborization in rat sympathetic neurons by blocking RhoA activationJournal of Neurochemistry2009ISSN 0022-3042
Stirling, Lee et al.Dual roles for RhoA/Rho-kinase in the regulated trafficking of a voltage-sensitive potassium channelMolecular Biology of the Cell2009ISSN 1059-1524
Martinelli, Roberta et al.ICAM-1-mediated endothelial nitric oxide synthase activation via calcium and AMP-activated protein kinase is required for transendothelial lymphocyte migrationMolecular Biology of the Cell2009ISSN 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/AktJournal of Biological Chemistry2009ISSN 0021-9258
Groysman, Maya et al.A negative modulatory role for rho and rho-associated kinase signaling in delamination of neural crest cellsNeural Development2008ISSN 1749-8104
Kirchner, Marieluise et al.Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenesMolecular Microbiology2008ISSN 0950-382X
Kanada, Masamitsu et al.Novel functions of Ect2 in polar lamellipodia formation and polarity maintenance during "contractile ring-independent" cytokinesis in adherent cellsMolecular Biology of the Cell2008ISSN 1059-1524
Navarro, Angels et al.T1α/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cellsAmerican Journal of Physiology - Lung Cellular and Molecular Physiology2008ISSN 1040-0605
Rupp, Paul A. et al.A role for RhoA in the two-phase migratory pattern of post-otic neural crest cellsDevelopmental Biology2007ISSN 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 tservice@cytoskeleton.com.