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Rac Pull-Down Activation Assay Kit

Rac Pull-Down Activation Assay Kit
5/5

$737.00

Cat.#:  80501

   Size:   30 Assays

In Stock

          Product Description          

Rac Pull-Down Activation Assay Kit

Cat. # 80501

Introduction

A. Background
Small GTPases are a super-family of cellular signaling regulators. Rac belongs to the Rho sub-family of GTPases that regulate cell motility, cell division, and gene transcription. GTP binding increases the activity of Rac, and the hydrolysis of GTP to GDP renders it inactive.
Currently the activation of Rac proteins is assayed with the binding of GTP-bound Rac to the p21-binding domain (PBD) of p21-activated protein kinase (PAK). This method is based on the observation that the active, GTP-bound Rac could bind to the PBD of PAK. However, the reproducibility of this method is poor. This is partially due to the relatively quick hydrolysis of GTP to GDP during the assay procedure, and the low binding affinity of PBD to Rac-GTP.
The Rac Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Rac-GTP, but not Rac-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a much shorter time. This assay provides the reliable results with consistent reproducibility.
These anti-Rac-GTP monoclonal antibody can also be used to monitor the activation of Rac in cells and in tissues by immunohistochemistry.
B. Assay Principle
The Rac Activation Assay Kit uses configuration-specific anti-Rac-GTP Mouse monoclonal antibody to measure Rac-GTP levels in cell extracts or in vitro GTPγS loading Rac activation assays. Anti-Rac-GTP mouse monoclonal antibody is first incubated with cell lysates containing Rac-GTP. Next, the GTP-bound Rac is pulled down by protein A/G agarose. Finally, the precipitated Rac-GTP is detected through immunoblot analysis using Anti-Rac Rabbit Polyclonal Antibody.
The anti-Rac-GTP monoclonal antibody can also be used to monitor the activation of Rac in cells and in tissues by immunohistochemistry.
C. Kit Components
1. Anti-Rac-GTP Mouse Monoclonal Antibody (Cat. # 26903): 30 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Rac-GTP from all vertebrates.
2. Protein A/G Agarose (Cat. # 30301): 600 µL of 50% slurry.
3. 5X Assay/Lysis Buffer (Cat. # 30302): 30 mL of 250 mM Tris-HCl, pH 8, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.
4. Anti-Rac Rabbit Polyclonal Antibody (Cat. # 21003): 50 µL (1mg/mL) in PBS, pH 7.4, contained 50% glycerol.
5. 100X GTPγS (Cat. # 30303): 50 µl at 10 mM, use 5 µL of GTPγS for  GTP-labeling of 0.5 mL of cell lysate.
6. 100X GDP (Cat. # 30304): 50 µl at 100 mM, use 5 µL of GDP for GDP-labeling of 0.5 mL of cell lysate.
7. HRP-Goat Anti-Rabbit IgG (Cat. #29002): 50 µL (0.4 mg/mL) in PBS, pH 7.4, contained 50% glycerol.
D. Materials Needed but Not Supplied
1. Stimulated and non-stimulated cell lysates
2. Protease inhibitors
3. 4 °C tube rocker or shaker
4. 0.5 M EDTA at pH 8.0
5. 1.0 M MgCl2
6. 2X reducing SDS-PAGE sample buffer
7. Electrophoresis and immunoblotting systems
8. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05%  Tween-20)
9. Immunoblotting blocking buffer (TBST containing 5% Non-fat Dry Milk or 3% BSA)
10. ECL Detection Reagents
E. Example Results
The following figure demonstrates example results seen with the Rac Activation Assay Kit. For reference only.
Rac Activation Assay Kit Rac Activation Assay. MEF cells were treated with (lane 2) or without (lane 1) PDGF. Cell lysates were incubated with an anti-Rac-GTP monoclonal antibody (Cat. # 26903) (top panel). The precipitated active Rac was immunoblotted with an anti-Rac rabbit polyclonal antibody (Cat # 21003). The bottom panel shows the Western blot with anti-Rac of the cell lysates used (5% of that used in the top panel).

Assay Procedure

A. Reagent Preparation
1X Assay/Lysis Buffer: Mix the 5X Stock (Cat. # 30302) briefly and dilute with deionized water to make 1X buffer. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin.
B. Sample Preparation
Adherent Cells
1. Culture cells (one 10-cm plate, ~107 cells) to approximately 80-90% confluence. Stimulate the cells with activator or inhibitor as desired.
2. Aspirate the culture media and wash twice with ice-cold PBS.
3. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cells (0.5-1 mL per 10 cm tissue culture plate).
4. Place the culture plates on ice for 10-20 minutes.
5. Detach the cells from the plates by scraping with a cell scraper.
6. Transfer the lysates to appropriate size tubes and place on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store the sample (~1-2 mg of total protein) on ice for immediate use, or snap freeze and store at -70°C for future use.
Suspension Cells
1. Culture cells and stimulate with activator or inhibitor as desired.
2. Perform a cell count and then pellet the cells through centrifugation.
3. Aspirate the culture media and wash twice with ice-cold PBS.
4. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cell pellet (0.5-1 mL per 107 cells).
5. Lyse the cells by repeated pipetting.
6. Transfer the lysates to appropriate size tubes and place them on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store sample on ice for immediate use, or snap freeze and store at -70°C for future use.
C. In vitro GTPγS/GDP Protein for Positive and Negative controls
Note: In vivo stimulation of cells will activate approximately 10% of the available Rac, whereas in vitro GTPγS protein loading will activate nearly 90% of Rac.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Rac protein) into two microcentrifuge tubes.
2. To each tube, add 20 µL of 0.5 M EDTA (final concentration of 20 mM).
3. Add 5 µL of 100 X GTPγS (Cat. # 30303) to the first tube as a positive control.
4. Add 5 µL of 100 X GDP (Cat. # 30304) to the second tube as a negative control.
5. Incubate both tubes at 30°C for 30 minutes with agitation.
6. Stop loading by placing the tubes on ice and adding 32.5 µL of 1 M MgCl2 (final concentration of 60 mM).
D. Affinity Precipitation of Activated G Protein
1. Aliquot 0.5-1 mL of cell lysates (about 1 mg of total cellular protein) to a microcentrifuge tube.
2. Adjust the volume to 1 mL with 1X Assay/Lysis Buffer (See Reagent Preparation).
3. Add 1 µL anti-Rac-GTP antibody (Cat. # 26903).
4. Prepare the protein A/G Agarose bead slurry (Cat. # 30301) by resuspending through vertexing or titrating.
5. Quickly add 20 µL of resuspended bead slurry to above tube.
6. Incubate the tube at 4°C for 1 hour with gentle agitation.
7. Pellet the beads through centrifugation at 5,000 x g for 1 min.
8. Aspirate and discard the supernatant (making sure not to disturb or remove the bead pellet).
9. Wash the beads 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each time.
10. After the third wash, pellet the beads through centrifugation and carefully remove all the supernatant.
11. Resuspend the bead pellet in 20 µL of 2X reducing SDS- PAGE sample buffer.
12. Boil the sample for 5 minutes.
13. Centrifuge it at 5,000 x g for 10 seconds.
E. Western Blot Analysis
1. Load 15 µL/well of pull-down supernatant to a polyacrylamide gel (17%). It is recommended to include a pre-stained MW standard (as an indicator of a successful transfer in step 3 below).
2. Perform SDS-PAGE following the manufacturer’s instructions.
3. Transfer the gel proteins to a PVDF or nitrocellulose membrane following the manufacturer’s instructions.
Note: Steps 4-11 are at room temperature with agitation
4. Following electroblotting, immerse the PVDF membrane in 100% Methanol for 15 seconds, and then allow it to dry at room temperature for 5 minutes.
Note: If Nitrocellulose is used instead of PVDF, step 4 Should be skipped.
5. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature with constant agitation.
6. Wash the blotted membrane three times with TBST, 5 minutes each time.
7. Incubate the membrane with Anti-Rac Rabbit Polyclonal Antibody (Cat. # 21003), which is freshly diluted 1:50~500 (depending on the amount of Rac proteins in your sample) in 5% non-fat dry milk or 3% BSA in TBST, for 1-2 hr at room temperature with constant agitation or at 4°C overnight.
8. Wash the blotted membrane three times with TBST, 5 minutes each time.
9. Incubate the membrane with a secondary antibody (Cat. # 29002), which is freshly diluted 1:1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 hr at room temperature with constant agitation.
10. Wash the blotted membrane three times with TBST, 5 minutes each time.
11. Use the detection method of your choice such as ECL.
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Stem Cells Dev. 2015 PMID:25209090
132. LPA-mediated migration of ovarian cancer cells involves translocalization of Gαi2 to invadopodia and association with Src and β-pix
Cancer Lett. 2015 PMID:25451317
133. Semaphorin-3F suppresses the stemness of colorectal cancer cells by inactivating Rac1
Cancer letters. 2015 PMID:25529012
134. Rac1/Pak1/p38/MMP-2 axis regulates angiogenesis in ovarian cancer
Clin Cancer Res. 2015 PMID:25595279
135. Inhibition of Rac1 Activity in the Hippocampus Impairs the Forgetting of Contextual Fear Memory
Mol Neurobiol. 2015 PMID:25613020
136. Neuronal apoptosis induced by selective inhibition of Rac GTPase versus global suppression of Rho family GTPases is mediated by alterations in distinct mitogen-activated protein kinase
J Biol Chem. 2015 PMID:25666619
137. Fibroblastic Transformation of Corneal Keratocytes by Rac Inhibition is Modulated by Extracellular Matrix Structure and Stiffness
J Funct Biomater. 2015 PMID:25874856
138. Qian Yang Yu Yin Granule-containing serum inhibits angiotensin II-induced proliferation. reactive oxygen species production. and inflammation in human mesangial cells via an NADPH oxidase 4-dependent pathway
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139. TIPE2 protein prevents injury-induced restenosis in mice
Biochim Biophys Acta. 2015 PMID:25912734
140. Dishevelled-2 regulates cocaine-induced structural plasticity and Rac1 activity in the nucleus accumbens
Neurosci Lett. 2015 PMID:25957559
141. A bibenzyl from Dendrobium ellipsophyllum inhibits migration in lung cancer cells
J Nat Med. 2015 PMID:26109451
142. The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice
Physiol Rep. 2015 PMID:26116550
143. A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration
Nat Commun. 2015 PMID:26166433
144. Uterine RAC1 via Pak1-ERM signaling directs normal luminal epithelial integrity conducive to on-time embryo implantation in mice
Cell Death Differ. 2015 PMID:26184908
145. Truncating mutation in the autophagy gene UVRAG confers oncogenic properties and chemosensitivity in colorectal cancers
Nat Commun. 2015 PMID:26234763
146. Rac1 contributes to cerebral ischemia reperfusion-induced injury in mice by regulation of Notch2
Neuroscience. 2015. PMID:26299339
147. Rac1 Regulates Endometrial Secretory Function to Control Placental Development
PLoS Genet. 2015 PMID:26305333
148. Sur8/Shoc2 promotes cell motility and metastasis through activation of Ras-PI3K signaling
Oncotarget. 2015 PMID:26384305
149. Rac1 augments Wnt signaling by stimulating beta-catenin-LEF-1 complex assembly independent of beta-catenin nuclear import
J Cell Sci. 2015 PMID:26403202
150. Rap1 GTPase Inhibits Tumor Necrosis Factor-à-Induced Choroidal Endothelial Migration via NADPH Oxidase- and NF-?B-Dependent Activation of Rac1
Am J Pathol. 2015 PMID:26476350
151. Cyclic Mechanical Loading Is Essential for Rac1-Mediated Elongation and Remodeling of the Embryonic Mitral Valve
Current Biology. 2015 PMID:26725196
152. Immunofluorescence and Confocal Microscopy of Neutrophils
Methods Mol Biol. 2014 PMID:18453118
153. Jak3 Enables Chemokine-Dependent Actin Cytoskeleton Reorganization by Regulating Cofilin and Rac/Rhoa GTPases activation
PLoS One. 2014 PMID:24498424
154. High incidence of naevi-associated BRAF wild-type melanoma and dysplastic naevi under treatment with the class I BRAF inhibitor vemurafenib
Acta Derm Venereol. 2014 PMID:24531394
155. A link between the nuclear-localized srGAP3 and the SWI/SNF chromatin remodeler Brg1
Molecular and Cellular Neuroscience. 2014 PMID:24561795
156. Phosphorylation of α-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells
Cytoskeleton. 2014 PMID:24574051
157. SHP2 Phosphatase Promotes Mast Cell Chemotaxis toward Stem Cell Factor via Enhancing Activation of the Lyn/Vavac Signaling Axis
The Journal of Immunology. 2014 PMID:24733849
158. ROBO1. a tumor suppressor and critical molecular barrier for localized tumor cells to acquire invasive phenotype: Study in African-American and Caucasian prostate cancer models
International Journal of Cancer. 2014 PMID:24752651
159. Transcription factors NRF2 and nf-kb are coordinated effectors of the RHO family. GTP binding protein rac1 during inflammation
J Biol Chem. 2014 PMID:24759106
160. PI(4.5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site
Development. 2014 PMID:24821989
161. Loss of ARHGDIA expression is associated with poor prognosis in HCC and promotes invasion and metastasis of HCC cells
Int J Oncol. 2014 PMID:24859471
162. srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons by inactivating RAC1
Asian Pac J Trop Med. 2014 PMID:25149377
163. Canonical and Non-canonical G-protein Signaling Helps Coordinate Actin Dynamics to Promote Macrophage Phagocytosis of Zymosan
Mol Cell Biol. 2014 PMID:25225330
164. p53 Down Regulates PDGF-Induced Formation of Circular Dorsal Ruffles in Rat Aortic Smooth Muscle Cells
PLoS One. 2014 PMID:25247424
165. TRPM2 mediates ischemic kidney injury and oxidant stress through RAC1
J Clin Invest. 2014 PMID:25295536
166. Triclosan Potentiates Epithelial-To-Mesenchymal Transition in Anoikis-Resistant Human Lung Cancer Cells
PLoS One. 2014 PMID:25329306
167. cAMP controls the restoration of endothelial barrier function after thrombin‐induced hyperpermeability via Rac1 activation
Physiological Reports. 2014 PMID:25344477
168. Cisplatin at Sub-toxic Levels Mediates Integrin Switch in Lung Cancer Cells
Anticancer Research. 2014 PMID:25503138
169. Geometry sensing through POR1 regulates Rac1 activity controlling early osteoblast differentiation in response to nanofiber diameter
Integr Biol (Camb). 2014 PMID:25539497
170. Neuregulin Mediates F-actin-driven Cell Migration through Inhibition of Protein Kinase D1 via Rac1 Protein
J Biol Chem. 2013 PMID:23148218
171. Rac1/PAK1 signaling promotes epithelial-mesenchymal transition of podocytes in vitro via triggering beta-catenin transcriptional activity under high glucose conditions
Int J Biochem Cell Biol. 2013 PMID:23153508
172. Reactive Oxygen Species Are Induced by Kaposi’s Sarcoma-Associated Herpesvirus Early during Primary Infection of Endothelial Cells To Promote Virus Entry
J Virol. 2013 PMID:23175375
173. Essential Role of Class II Phosphatidylinositol-3-kinase-C2alpha in Sphingosine 1-Phosphate Receptor-1-mediated Signaling and Migration in Endothelial Cells
J Biol Chem. 2013 PMID:23192342
174. PAK-PIX interactions regulate adhesion dynamics and membrane protrusion to control neurite outgrowt
J Cell Sci. 2013 PMID:23321640
175. Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization. Macrophage Phagocytosis. and Cytokine-Stimulated Neutrophil Recruitment
PLoS One. 2013 PMID:23383154
176. Myocardial Rac1 exhibits partial involvement in thyroxin-induced cardiomyocyte hypertrophy and its inhibition is not sufficient to improve cardiac dysfunction or contractile abnormalities in mouse papillary muscles
J Cardiovasc Pharmacol. 2013 PMID:23429587
177. A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates
Autophagy. 2013 PMID:23439251
178. Rac1 is required for matrix metalloproteinase-13 production by chondrocytes in response to fibronectin fragments
Arthritis Rheum. 2013 PMID:23460186
179. Lis1 mediates planar polarity of auditory hair cells through regulation of microtubule organization
Development. 2013 PMID:23533177
180. Fer Protein-Tyrosine Kinase Promotes Lung Adenocarcinoma Cell Invasion and Tumor Metastasis
Mol Cancer Res. 2013 PMID:23699534
181. Pilus Phase Variation Switches Gonococcal Adherence to Invasion by Caveolin-1-Dependent Host Cell Signaling
PLoS Pathog. 2013 PMID:23717204
182. ArhGAP15. a Rac-specific GTPase-activating Protein. Plays a Dual Role in Inhibiting Small GTPase Signaling
J Biol Chem. 2013 PMID:23760270
183. Kalirin-7 Mediates Cocaine-Induced AMPA Receptor and Spine Plasticity. Enabling Incentive Sensitization
J Neurosci. 2013 PMID:23825406
184. p21-Activated Kinase (PAK) Regulates Cytoskeletal Reorganization and Directional Migration in Human Neutrophils
PLoS One. 2013 PMID:24019894
185. Loss of TAK1 increases cell traction force in a ROS-dependent manner to drive epithelial–mesenchymal transition of cancer cells
Cell Death Dis. 2013 PMID:24113182
186. A directional switch of integrin signaling and a new anti-thrombotic strategy
Nature. 2013 PMID:24162846
187. JAK tyrosine kinases promote hierarchical activation of Rho and Rap modules of integrin activation
J Cell Biol. 2013 PMID:24368807
188. he cooperative action of bacterial fibronectin-binding proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling
Cell Microbiol. 2012 PMID:21999233
189. Interferon gamma induces actin polymerization. Rac1 activation and down regulates phagocytosis in human monocytic cells
Cytokine. 2012 PMID:22137120
190. Co-regulation of transcellular and paracellular leak across microvascular endothelium by dynamin and Rac
Am J Pathol. 2012 PMID:22203054
191. Association of syntenin-1 with M-RIP polarizes Rac-1 activation during chemotaxis and immune interactions
J Cell Sci. 2012 PMID:22349701
192. rac1 is essential in cocaine-induced structural plasticity of nucleus accumbens neurons
Nat Neurosci. 2012 PMID:22522400
193. Antagonistic activities of Rho and Rac GTPases underlie the transition from neural crest delamination to migration
Dev Dyn. 2012 PMID:22553120
194. miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling
EMBO J. 2012 PMID: 22588079
195. Interferon-beta Therapy Against EAE Is Effective Only When Development of the Disease Depends on the NLRP3 Inflammasome
Sci Signal. 2012 PMID:22623753
196. Rac1 inhibition prevents tissue contraction and MMP mediated matrix remodeling in the conjunctiva
Invest Ophthalmol Vis Sci. 2012 PMID:22695959
197. Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex
J Cell Biol. 2012 PMID:22891260
198. Rac-induced left ventricular dilation in thyroxin-treated ZmRacD transgenic mice: role of cardiomyocyte apoptosis and myocardial fibrosis
PLoS One. 2012 PMID:22936985
199. Stimulation of cortical myosin phosphorylation by p114RhoGEF drives cell migration and tumor cell invasion
PLoS One. 2012 PMID:23185572
200. Rac1 controls Schwann cell myelination through cAMP and NF2/merlin
J Neurosci. 2012 PMID:23197717
201. Nerve growth factor-induced formation of axonal filopodia and collateral branches involves the intra-axonal synthesis of regulators of the actin-nucleating Arp2/3 complex
J Neurosci. 2012 PMID:23223289
202. Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation
Am J Physiol Heart Circ Physiol. 2011 PMID:21037229
203. K-Ras mediated murine epidermal tumorigenesis is dependent upon and associated with elevated Rac1 activity
PLoS One. 2011 PMID:21358804
204. Asymmetric Mbc. active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion in Drosophila
Development. 2011 PMID:21389053
205. Aspect ratio determines the quantity of mesoporous silica nanoparticle uptake by a small GTPase-dependent macropinocytosis mechanism
ACS Nano. 2011 PMID:21563770
206. R-Ras and Rac GTPase Cross-talk Regulates Hematopoietic Progenitor Cell Migration. Homing. and Mobilization
J Biol Chem. 2011 PMID:21572048
207. Differential roles of Smad2 and Smad3 in the regulation of TGF-beta1-mediated growth inhibition and cell migration in pancreatic ductal adenocarcinoma cells: control by Rac1
Mol Cancer. 2011 PMID:21624123
208. NADPH oxidase activation by hyperglycaemia in cardiomyocytes is independent of glucose metabolism but requires SGLT1
Cardiovasc Res. 2011 PMID:21859816
209. Latent KSHV infection increases the vascular permeability of human endothelial cells
Blood. 2011 PMID:21881052
210. Comparative RNAi screening identifies a conserved core metazoan actinome by phenotype
J Cell Biol. 2011 PMID:21893601
211. Rac1 and Stathmin but Not EB1 Are Required for Invasion of Breast Cancer Cells in Response to IGF-I
Int J Cell Biol. 2011 PMID:21961005
212. Ethanol increases p190RhoGAP activity. leading to actin cytoskeleton rearrangements
J Neurochem. 2011 PMID:21985251
213. Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis
J Leukoc Biol. 2010 PMID:20610796
214. Intracellular Ca2+ can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells
FEBS Lett. 2010 PMID:20621840
215. Antioxidants counteract nicotine and promote migration via RacGTP in oral fibroblast cells
J Periodontol. 2010 PMID:20636139
216. Physiological activation of synaptic Rac>PAK (p-21 activated kinase) signaling is defective in a mouse model of fragile X syndrome
J Neurosci. 2010 PMID:20720104
217. SUMOylation of the GTPase Rac1 is required for optimal cell migration
Nat Cell Biol. 2010 PMID:20935639