Product Uses Include
Control for Rho, Rac and Cdc42 pathway activation
Study the effects of Rho family small G-protein activation on other signaling pathways
Study the cell-type specific cross-talk between signaling pathways for Rho, Rac and Cdc42
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).
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).
|Nath, Anu S. et al.||Modulation of the cell membrane lipid milieu by peroxisomal β-oxidation induces Rho1 signaling to trigger inflammatory responses||Cell Reports||2022||ISSN 2211-1247|
|Jayabal, Panneerselvam et al.||NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growth||Cell Reports||2021||ISSN 2211-1247|
|Merlini, Mario et al.||Microglial Gi-dependent dynamics regulate brain network hyperexcitability||Nature Neuroscience||2021||ISSN 1546-1726|
|Rong, Zhouyi et al.||Activation of FAK/Rac1/Cdc42-GTPase signaling ameliorates impaired microglial migration response to Aβ42 in triggering receptor expressed on myeloid cells 2 loss-of-function murine models||FASEB Journal||2020||ISSN 1530-6860|
|Chen, Zihang et al.||Distinct roles of srGAP3-Rac1 in the initiation and maintenance phases of neuropathic pain induced by paclitaxel||The Journal of physiology||2020||ISSN 1469--7793|
|Larribère, Lionel et al.||NF1-RAC1 axis regulates migration of the melanocytic lineage||Translational Oncology||2020||ISSN 1936-5233|
|Andrews, Madeline G. et al.||Mtor signaling regulates the morphology and migration of outer radial glia in developing human cortex||eLife||2020||ISSN 2050-084X|
|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|
|Matsuda, Jun et al.||ARHGEF7 (β-PIX) is required for the maintenance of podocyte architecture and glomerular function||Journal of the American Society of Nephrology||2020||ISSN 1533-3450|
|García-Ponce, Alexander et al.||Epac1 Is Crucial for Maintenance of Endothelial Barrier Function through A Mechanism Partly Independent of Rac1||Cells||2020||ISSN 2073-4409|
|Kolyvushko, Oleksandr et al.||Equine alphaherpesviruses require activation of the small gtpases rac1 and cdc42 for intracellular transport||Microorganisms||2020||ISSN 2076-2607|
|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|
|Costa, Joana Freitas et al.||The Role of Rac GTPase in Dendritic Spine Morphogenesis and Memory||Frontiers in Synaptic Neuroscience||2020||ISSN 1663-3563|
|Li, Ling Zhu et al.||Rac1 Modulates Excitatory Synaptic Transmission in Mouse Retinal Ganglion Cells||Neuroscience Bulletin||2019||ISSN 1995-8218|
|Rizzi, Caterina et al.||NGF steers microglia toward a neuroprotective phenotype||Glia||2018||ISSN 1098-1136|
|Jackson, Edwin K. et al.||8-aminoguanine induces diuresis, natriuresis, and glucosuria by inhibiting purine nucleoside phosphorylase and reduces potassium excretion by inhibiting Rac1||Journal of the American Heart Association||2018||ISSN 2047-9980|
|Kimura-Yoshida, Chiharu et al.||Cytoplasmic localization of GRHL3 upon epidermal differentiation triggers cell shape change for epithelial morphogenesis||Nature Communications||2018||ISSN 2041-1723|
|Robins, Richard et al.||Rac1 activation in podocytes induces the spectrum of nephrotic syndrome||Kidney International||2017||ISSN 1523-1755|
|Jones, Eleanor L. et al.||Dendritic Cell Migration and Antigen Presentation Are Coordinated by the Opposing Functions of the Tetraspanins CD82 and CD37||The Journal of Immunology||2016||ISSN 0022--1767|
|Jiang, Lizhu et al.||Inhibition of Rac1 Activity in the Hippocampus Impairs the Forgetting of Contextual Fear Memory||Molecular Neurobiology||2016||ISSN 1559-1182|
|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|
|Reinardy, Jessica L. et al.||Phosphorylation of threonine 794 on Tie1 by Rac1/PAK1 reveals a novel angiogenesis regulatory pathway||PLoS ONE||2015||ISSN 1932-6203|
|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|
|Staiculescu, Marius C. et al.||Prolonged vasoconstriction of resistance arteries involves vascular smooth muscle actin polymerization leading to inward remodelling||Cardiovascular Research||2013||ISSN 0008-6363|
|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|
|Rom, Slava et al.||Glycogen synthase kinase 3β inhibition prevents monocyte migration across brain endothelial cells via Rac1-GTPase suppression and down-regulation of active integrin conformation||American Journal of Pathology||2012||ISSN 0002-9440|
|Schmidt, Gudula et al.||Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1||Nature 1997 387:6634||1997||ISSN 1476--4687|
|Flatau, Gilles et al.||Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine||Nature||1997||ISSN 0028--0836|
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.
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