Product Uses Include
Exoenzyme C3 transferase is an ADP ribosyl transferase that selectively ribosylates RhoA, RhoB and RhoC proteins on asparagine residue 41, rendering them inactive. It has extremely low affinity for other members of the Rho family such as Cdc42 and Rac1 and does therefore not affect these GTPases. Hence, C3 transferase is a very potent and useful reagent to specifically block RhoA/B/C signaling.
C3 transferase has been produc ed by expression in E. coli as a His-tagged protein. The recombinant protein is 24 kDa in size and is supplied as a lyophilized powder. Reconstitution of the protein in water to 1 mg/ml leaves the protein in the following buffer: 20 mM Tris pH 7.5, 50 mM NaCl, 0.5% sucrose and 0.1% dextran. Protein concentration is determined by the Precision Red Advanced Protein Assay Reagent Cat. # ADV02.
C3 transferase protein is also available in a cell permeable format (Cat. CT04) for fast, efficient and simple inhibition of RhoA/B/C in living cells.
Purity is determined by scanning densitometry of protein run on SDS-PAGE gels. CT03 consists of more than 90% pure exoenzyme C3 Transferase.
Figure 1: Exoenzyme C3 transferase purity determination. A 20 µg sample of CT03 was separated by SDS-PAGE and protein was stained with coomassie blue. Protein quantitation was performed using Precision Red Assay reagent (Cat. # ADV02). Purity was determined by scanning densitometry. The protein was determined to be >90% pure.
Biological activity of C3 transferase is verified by the ability of the protein to ribosylate RhoA protein in platelet lysates in vitro (Fig. 2).
Figure 1: ADP-ribosylation of RhoA protein in human platelet extract. Platelet extract (100 µg) was reacted with C3 protein (1 µg) for 30 min at 37°C. Extracts were run on non-denaturing gel electrophoresis and RhoA protein was detected by Western blot. Lane 1 shows untreated extracts. Lane 2 shows C3 transferase treated extracts. The RhoA in the C3 transferase treated extract shows increased migration in the gel due to its ADP ribosylation.
|Table 1. Examples of how Cytoskeleton, Inc's C3 transferase has been used on cells to inactivate RhoA.|
Method of introduction into cells
Concentration of C3 used (mg/ml)
added to culture media
Primary Aplysia bag cells
|Du, Xing et al.||The mevalonate pathway promotes the metastasis of osteosarcoma by regulating YAP1 activity via RhoA||Genes & Diseases||2022|
|McCray, Brett A. et al.||Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extension||Nature Communications||2021||ISSN 2041-1723|
|Landino, Jennifer et al.||Rho and F-actin self-organize within an artificial cell cortex||Current Biology||2021||ISSN 1879-0445|
|Xie, Lang et al.||MYO1B enhances colorectal cancer metastasis by promoting the F-actin rearrangement and focal adhesion assembly via RhoA/ROCK/FAK signaling||Annals of Translational Medicine||2021||ISSN 2305-5839|
|Zhou, Qun et al.||Inflammatory Immune Cytokine TNF-α Modulates Ezrin Protein Activation via FAK/RhoA Signaling Pathway in PMVECs Hyperpermeability||Frontiers in Pharmacology||2021||ISSN 1663-9812|
|Lu, Jiaoyang et al.||Basement Membrane Regulates Fibronectin Organization Using Sliding Focal Adhesions Driven by a Contractile Winch||Developmental Cell||2020||ISSN 1878-1551|
|Evans, Frances et al.||Signaling pathways in cytoskeletal responses to plasma membrane depolarization in corneal endothelial cells||Journal of Cellular Physiology||2020||ISSN 1097-4652|
|Pal, Debadrita et al.||Rac and Arp2/3-Nucleated Actin Networks Antagonize Rho During Mitotic and Meiotic Cleavages||Frontiers in Cell and Developmental Biology||2020||ISSN 2296-634X|
|Wang, Lei et al.||YAP and TAZ protect against white adipocyte cell death during obesity||Nature Communications||2020||ISSN 2041-1723|
|Miao, Hui et al.||Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constriction||Journal of Cell Biology||2019||ISSN 1540-8140|
|Zahra, Fatema Tuz et al.||Endothelial RhoA GTPase is essential for in vitro endothelial functions but dispensable for physiological in vivo angiogenesis||Scientific Reports||2019||ISSN 2045-2322|
|Ko, Clint S. et al.||Microtubules promote intercellular contractile force transmission during tissue folding||Journal of Cell Biology||2019||ISSN 1540-8140|
|Hübner, Kathleen et al.||Wnt/β-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling||Nature Communications||2018||ISSN 2041-1723|
|Raya-Sandino, Arturo et al.||Zonula occludens-2 regulates Rho proteins activity and the development of epithelial cytoarchitecture and barrier function||Biochimica et Biophysica Acta - Molecular Cell Research||2017||ISSN 1879-2596|
|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|
|Ren, Yu et al.||Induction of cell cycle arrest by increasing GTP-RhoA levels via taxol-induced microtubule polymerization in renal cell carcinoma||Molecular Medicine Reports||2017||ISSN 1791-3004|
|Song, Nan et al.||NLRP3 Phosphorylation Is an Essential Priming Event for Inflammasome Activation||Molecular Cell||2017||ISSN 1097-4164|
|Mason, Frank M. et al.||RhoA GTPase inhibition organizes contraction during epithelial morphogenesis||Journal of Cell Biology||2016||ISSN 1540-8140|
|Park, Yong Hwan et al.||Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS||Nature Immunology||2016||ISSN 1529-2916|
|Tivari, Samir et al.||An in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: A tool for molecular mechanism studies and hypothesis generation||Journal of Visualized Experiments||2015||ISSN 1940-087X|
|Lemons, M L et al.||Integrins and cAMP mediate netrin-induced growth cone collapse||Brain Research||2013||Article Link|
|Takefuji, Mikito et al.||RhoGEF12 controls cardiac remodeling by integrating G protein- and integrindependent signaling cascades||Journal of Experimental Medicine||2013||ISSN 0022-1007|
|Takefuji, Mikito et al.||G13-mediated signaling pathway is required for pressure overload-induced cardiac remodeling and heart failure||Circulation||2012||ISSN 0009-7322|
|Wong, Hon Kit et al.||Merlin/NF2 regulates angiogenesis in schwannomas through a Rac1/semaphorin 3F-dependent mechanism||Neoplasia||2012||ISSN 1476-5586|
|Melendez, Jaime et al.||RhoA GTPase Is Dispensable for Actomyosin Regulation but Is Essential for Mitosis in Primary Mouse Embryonic Fibroblasts * □ S||2011||Article Link|
|Marshall, Andrew K. et al.||ERK1/2 signaling dominates over RhoA signaling in regulating early changes in RNA expression induced by endothelin-1 in neonatal rat cardiomyocytes||PLoS ONE||2010||ISSN 1932-6203|
|Oblander, Samantha A. et al.||Distinct PTPmu-associated signaling molecules differentially regulate neurite outgrowth on E-, N-, and R-cadherin||Molecular and Cellular Neuroscience||2010||ISSN 1044-7431|
|Toullec, Aurore et al.||Oxidative stress promotes myofibroblast differentiation and tumour spreading||EMBO Molecular Medicine||2010||ISSN 1757-4684|
|Benink, Hélène A. et al.||Concentric zones of active RhoA and Cdc42 around single cell wounds||Journal of Cell Biology||2005||ISSN 0021-9525|
|O'neil, Caroline H et al.||Stimulation of Vascular Smooth Muscle Cell Proliferation and Migration by Apolipoprotein(a) Is Dependent on Inhibition of Transforming Growth Factor-Î² Activation and on the Presence of Kringle IV Type 9*||2004||Article Link|
|Simpson, Kaylene J. et al.||Functional analysis of the contribution of RhoA and RhoC GTPases to invasive breast carcinoma||Cancer Research||2004||ISSN 0008-5472|
|Ridley, Anne J.||Pulling Back to Move Forward||Cell||2004|
|Burakov, Anton et al.||Centrosome positioning in interphase cells||Journal of Cell Biology||2003||ISSN 0021-9525|
|Valderrama, Ferran et al.||The Golgi-associated COPI-coated buds and vesicles contain β/γ-actin||Proceedings of the National Academy of Sciences of the United States of America||2000||ISSN 0027-8424|
Question 1: What is the difference between the Rho inhibitors CT03 and CT04?
Answer 1: The only difference between these C3 Transferase proteins (Cat. # CT03 and CT04) is that CT04 is covalently linked to a proprietary cell penetrating moiety via a disulfide bond. In this way, CT04 is a much better reagent to use to inhibit Rho activity in living cells.
Question 2: How can I assess whether Rho activity is changing following CT03 treatment?
Answer 2: There are multiple ways to measure changes in Rho activity. If CT03 has been delivered to cells via micro-injection or pinocytic uptake, 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). You can also measure CT03’s ability to ADP ribosylate native Rho in human platelet extracts in vitro (please see the CT03 datasheet for more details). Briefly, a standard biological assay for monitoring the ADP ribosylation of Rho consists of an in vivo ribosylation reaction followed by non-denaturing gel electophoresis and Western blot analysis using Cytoskeleton’s anti-Rho monoclonal antibody (Cat. # ARH03). Stringent quality control ensures that >80% of native Rho protein is ADP ribosylated by the recombinant C3 transferase.
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