Product Uses Include
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Control for Rho, Rac and Cdc42 pathway activation
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Study the effects of Rho family small G-protein activation on other signaling pathways
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Study the cell-type specific cross-talk between signaling pathways for Rho, Rac and Cdc42
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Study the effects of Rho family small G-protein activation on the re-arrangement of the actin cytoskeleton
The active site of CN04 is based on the catalytic domain of the bacterial cytotoxic necrotizing factor (CNF) toxins. The catalytic domain is covalently attached to a proprietary cell penetrating moiety. CN04 directly activates Rho GTPase isoforms by deamidating glutamine-63 of Rho and glutamine-61 of Rac and Cdc42 in their respective Switch II regions (1,2). This modification converts glutamine-63 to glutamate, which blocks intrinsic and GAP-stimulated GTPase activity, resulting in constitutively active endogenous Rho, Rac and Cdc42 (3). CN04 robustly increases the level of GTP-bound RhoA, Rac1 and Cdc42 within 2-4 h after addition to the culture medium. CN04 can be used when a direct activator of Rho family proteins is required rather than a classic indirect activator (e.g., LPA, EGF, Bradykinin and Sphingosine-1-phosphate) that concomitantly activate other signaling pathways (e.g., Ras, PI3K and PLC).
References
1. Lerm M., et al. 1999. Deamidation of Cdc42 and Rac by Escherichia coli cytotoxic necrotizing factor 1: activation of c-Jun N-terminal kinase in HeLa cells. Infection and immunity. 67, 496-503.
2. Schmidt G., et al. 1997. Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature. 387, 725-729.
3. Flatau G., et al. 1997. Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature. 387, 729-733.
Above : Rac activation in Swiss 3T3 cells. F-actin is visualized with fluorescent green phalloidin staining (Cat.# PHDG1) and nuclear blue DNA staining with Dapi. Cells were activated with Cat. # CN04 (right).
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 attservice@cytoskeleton.com
Larribère, L. et al. NF1-RAC1 axis regulates migration of the melanocytic lineage. Transl. Oncol. 13, 100858 (2020).
Chen, Z. et al. Distinct roles of srGAP3‐Rac1 in the initiation and maintenance phases of neuropathic pain induced by paclitaxel. J. Physiol. 598, 2415–2430 (2020).
Rong, Z. et al. Activation of FAK/Rac1/Cdc42‐GTPase signaling ameliorates impaired microglial migration response to Aβ 42 in triggering receptor expressed on myeloid cells 2 loss‐of‐function murine models. FASEB J. 34, 10984–10997 (2020).
Larribère, L. et al. NF1-RAC1 axis regulates migration of the melanocytic lineage. Transl. Oncol. 13, 100858 (2020).
García-Ponce, A. et al. Epac1 Is Crucial for Maintenance of Endothelial Barrier Function through A Mechanism Partly Independent of Rac1. Cells 9, 2170 (2020).
Kolyvushko, O., Kelch, M. A., Osterrieder, N. & Azab, W. Equine Alphaherpesviruses Require Activation of the Small GTPases Rac1 and Cdc42 for Intracellular Transport. Microorganisms 8, 1013 (2020).
Rom, S. et al. Hyperglycemia and advanced glycation end products disrupt BBB and promote occludin and claudin-5 protein secretion on extracellular microvesicles. (2020) doi:10.1038/s41598-020-64349-x.
Li, L. Z. et al. Rac1 Modulates Excitatory Synaptic Transmission in Mouse Retinal Ganglion Cells. Neurosci. Bull. 35, 673–687 (2019).
Rizzi, C. et al. NGF steers microglia toward a neuroprotective phenotype. Glia 66, 1395–1416 (2018).
Jackson, E. K., Mi, Z., Kleyman, T. R. & Cheng, D. 8-aminoguanine induces diuresis, natriuresis, and glucosuria by inhibiting purine nucleoside phosphorylase and reduces potassium excretion by inhibiting Rac1. J. Am. Heart Assoc. 7, (2018).
Schmidt G., et al. 1997. Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1.
Nature. 387, 725-729. Flatau G., et al.
1997. Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature. 387, 729-733.
Question 1: Can the direct Rho/Rac/Cdc42 activator CN04 be used with cells growing in culture?
Answer 1: Yes, CN04 is specifically designed to be used as a Rho/Rac/Cdc42 activator with cultured cells. The active site of CN04 is based on the catalytic domain of the bacterial cytotoxic necrotizing factor (CNF) toxins. The catalytic domain of CN04 is covalently attached to a proprietary cell penetrating moiety. Upon entry into the cell, CN04 directly activates Rho GTPase isoforms by deamidating glutamine-63 of Rho and glutamine-61 of Rac and Cdc42 in their respective Switch II regions. This modification converts glutamine-63 to glutamate, which blocks intrinsic and GAP-stimulated GTPase activity, resulting in constitutively active endogenous Rho, Rac and Cdc42. CN04 robustly increases the level of GTP-bound RhoA, Rac1 and Cdc42 within 2-4 h after addition to the culture medium.
Question 2: How can I assess whether Rho, Rac and/or Cdc42 activity is changing in my cells following CN04 treatment?
Answer 2: There are multiple ways to measure changes in Rho, Rac and Cdc42 activity. To visualize a change in a cell’s cytoskeleton mediated by Rho family proteins, we recommend examining stress fiber formation and edge ruffling with fluorescently-labeled phalloidin (Cat. # PHDG1, PHDH1, PHDN1, PHDR1). These Acti-stain phalloidins label F-actin-containing structures and fibers. Activation of Rho family proteins can be directly quantified with either our pull-down or G-LISA activation assays. For RhoA, use the BK036 pull-down or BK 124 G-LISA. For Rac1, use the BK035 pull-down or BK128 G-LISA. For Cdc42, use the BK034 pull-down or BK127 G-LISA.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.
Cytoskeleton公司成立于1993年，专注于生物化学和细胞过程研究中的纯化蛋白和便捷试剂盒开发与生产。公司提供药物筛选、信号转导、细胞骨架研究相关的系列试剂盒和产品，尤其以细胞骨架相关研究见长，既能满足于样品较少的科学研究，也可以用于小规模筛选研究和高通量大规模筛选研究。此外，公司还提供微管蛋白，肌动蛋白，小G蛋白，GAPs，GEFs等现有产品的药物筛选服务。
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