© 2007 Cell Signaling Technology, Inc. This productisfor Store at –20°C Species enclosed inparenthesesarepredicted toreactbasedon100% sequencehomology. Species Cross-Reactivity Key: Applications Key: Rock Axis. Astroglial PhenotypeviaDeactivationoftheRhoGTPase- John, G.R.etal.(2004)Interleukin-1 Fibroblasts. Essential fortheEnhancedContractilePhenotypeofFibrotic phoinositide 3-Kinase/Akt-dependentPathwayandIs blast InductionthroughtheETA Receptorviaarac/Phos Shi-Wen, X.etal.(2004)Endothelin-1PromotesMyofibro Selected ApplicationReferences: ity chromatography. ezrin. AntibodiesarepurifiedbyproteinAandpeptideaffin corresponding to the residues surrounding Thr567 of human by immunizing rabbits with a synthetic peptide (KLH-coupled) Source/Purification: merlin orband4.1. antibody doesnotcross-reactwithrelatedproteinssuchas detects endogenouslevelsofezrin,radixinandmoesin.The Specificity/Sensitivity: differentiation (6). Tyr353 signalduringepithelial ofezrintransmitsasurvival of residues upongrowthfactorstimulation.Phosphorylation attyrosine transformation (5).Ezrinisalsophosphorylated for cytoskeletalrearrangementsandoncogene-induced atThr567ofezrinisrequired tion (3,4).Phosphorylation key roleinregulatingERMproteinconformationandfunc amino- andcarboxy-terminalassociationmayplaya ezrin, Thr564ofradixin,Thr558moesin)disruptstheir tion atacarboxy-terminalthreonineresidue(Thr567of inactive cytosolicmonomersordimers(2).Phosphoryla their amino-andcarboxy-terminaldomains,existingas proteins undergointraorintermolecularinteractionbetween sion, membranerufflingandmicrovilliformation(1).ERM and theactincytoskeletonareinvolvedincelladhe proteins functionaslinkersbetweentheplasmamembrane Background:

0.1% Tween-20 IMPORTANT: ForWestern blots,incubatemembranewith dilutedantibodyin5%w/vBSA, 1X TBS, #3142 n n Antibody Ezrin/Radixin/Moesin Applications   in vitro (30 Western mini-blots) Large (10 Western mini-blots) Small J. Neurosci. Mol. Biol.Cell W The ezrin,radixinandmoesin(ERM) researchuseonlyandisnotintendedforinhumansoranimals. W at 4°Cwithgentle shaking,overnight. —Western 21 (11), 2837–2845. Application: W. Polyclonal antibodiesareproduced Ezrin/Radixin/Moesin Antibody 15,2707–2719.Application:W. H Species Cross-Reactivity* —human 300 µl 100 µl IP —Immunoprecipitation H, M,R,Mk,B b InducesaReactive M

—mouse R - —rat - IHC - - - - —Immunohistochemistry 80 kDaEzrinandRadixin. Hm

Background References: cells, usingEzrin/Radixin/MoesinAntibody. Western blotanalysisofextractsfromHeLa,L929,C6andCOS

(5)  (3)  (6)  (4)  (1)  (2)  —hamster 75 kDaMoesin. Molecular Wt. Tran Quang,C.etal.(2000) Matsui, T. etal.(1998) Gautreau, A.etal.(1999) Gautreau, A.etal.(2000) Tsukita, S.andYonemura, S.(1999) Mangeat, P. etal.(1999) 96, 7300–7305. 274, 34507–34510. 46.5 37.5 kDa 165 105 57 76 28 Mk —monkey HeLa IC —Immunocytochemistry

L929 Mi J. CellBiol. C6 rev. 11/07/07 —mink Trends CellBiol. Proc. Natl.Acad.Sci.USA J. CellBiol. Source Rabbit EMBO J. COS C 140,647–657. —chicken Ezrin/Radixin Moesin J. Biol.Chem. 19,4565–4576. 150,193–203. IF —Immunofluorescence 9,187–192. X —Xenopus

Western blotting Antibody #3141 Phospho-Ezrin (Thr567)/Radixin(Thr564)/Moesin(Thr558) Companion Products: Recommended AntibodyDilutions: 20X LumiGLO Biotinylated ProteinLadder#7727 #7720 Prestained ProteinMarker, BroadRange(PremixedFormat) Anti-rabbit IgG,HRP-linkedAntibody#7074 Moesin (Q480)Antibody#3150 Moesin Antibody#3146 (41A3) RabbitmAb#3149 Phospho-Ezrin (Thr567)/Radixin(Thr564)/Moesin(Thr558) Phospho-Ezrin (Tyr353) Antibody#3144 Ezrin Antibody#3145 *Species cross-reactivity is determined by Western blot. Do notaliquottheantibody NaCl, 100µg/mlBSAand50%glycerol.Storeat–20°C. Storage: Z —zebra fish F —Flow cytometry Support Orders Suppliedin10mMsodiumHEPES(pH7.5),150 Web ® B Reagentand20XPeroxide#7003 —bovine n n n E —ELISA www.cellsignal.com [email protected] 877-678-TECH (8324) [email protected] 877-616-CELL (2355) . All —all species expected D —DELFIA

® 1:1000

page  of 2 © 2007 Cell Signaling Technology, Inc.

molecular weights. to verifyelectrotransferandbiotinylatedproteinladder(#7727,10μl/lane)determine NOTE: A generalprotocolforsamplepreparationisdescribedbelow. B NOTE: A 1X TBS,0.1%Tween-20 at4°Cwithgentleshaking,overnight. For Western blots,incubatemembranewithdilutedantibodyin5%w/vBSA, #3142 8. 7. 6. 5. 4. 3. 2. 1. Protein Blotting 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. Solutions andReagents PreparesolutionswithMilli-Qorequivalentlypurifiedwater. CSTrecommendsloadingprestainedmolecularweightmarkers(#7720,10μl/lane)

Orders Primary AntibodyDilutionBuffer: Primary Blocking Buffer: 10X Tris BufferedSaline(TBS): 1X SDSSampleBuffer: Electrotransfer tonitrocelluloseorPVDFmembrane. Load 20μlontoSDS-PAGE gel(10cmx10cm). Microcentrifuge for5minutes. Heat a20μlsampleto95–100°Cfor5minutes;coolonice. Sonicate for10–15secondstoshearDNAandreducesampleviscosity. plate andtransfertheextracttoamicrocentrifugetube.Keeponice. 500 μlperplateof10cmdiameterplate).Immediatelyscrapethecellsoff Lyse cellsbyadding1XSDSsamplebuffer(100μlperwellof6-wellplateor Aspirate mediafromcultures;washcellswith1XPBS;aspirate. Treat cellsbyaddingfreshmediacontainingregulatorfordesiredtime. membranes, whichCSTrecommends.PVDFmembranesmayalsobeused. Blotting Membrane: Biotinylated ProteinLadderDetectionPack#7727 Prestained ProteinMarker, BroadRange(PremixedFormat)#7720 LumiGLO to horseradishperoxidase(HRP),anti-biotinantibodyconjugatedHRP, biotinylated proteinladder, anti-rabbit (#7074)antibodyconjugated secondary Phototope While stirring,add20μlTween-20 (100%). for 20ml,add2ml10XTBSto18water, mix.Add1.0gBSAandmixwell. Bovine SerumAlbumin(BSA) Wash Buffer: mix well.Whilestirring,add0.15mlTween-20 (100%). 150 ml,add15ml10XTBSto135water, milkand mix.Add7.5gnonfatdry Milk(weighttovolume[w/v]) Nonfat Dry base, 80gNaCl;adjustpHto7.6withHCl(useat1X). Transfer Buffer: 10% glycerol,50mMDTT, 0.01%w/vbromophenolblueorphenolred 1X PhosphateBufferedSaline(PBS) n Western AntibodyIncubationinBSA) ImmunoblottingProtocol (Primary ® 877-616-CELL (2355)[email protected] chemiluminescentreagentandperoxide. ® -HRP Western BlotDetectionSystem 1XTBS,0.1%Tween-20 (TBS/T) 25mMTris base,0.2Mglycine,20%methanol(pH8.5) 1XTBS,0.1%Tween-20 milk;for with5%w/vnonfatdry Thisprotocolhasbeenoptimizedfornitrocellulose 62.5mMTris-HCl (pH6.8at25°C),2%w/vSDS, To prepare1literof10XTBS:24.2gTris 1XTBS,0.1%Tween-20 with5%BSA; #7071 : Includes Support n

877-678-TECH (8324)[email protected] D branes, adjustvolumesaccordingly. NOTE: C following LumiGLO NOTE: NOTE: 1. Detection ofProteins 7. 6. 5. 4. 3. 2. 1. Membrane BlockingandAntibodyIncubations 2. Volumes arefor10cmx(100 Duetothekineticsofdetectionreaction,signalismostintenseimmediately LumiGLO

temperature. Peroxide and9.0mlMilli-Qwater)withgentleagitationfor1minuteatroom Incubate membranewith10mlLumiGLO Wash threetimesfor5minuteseachwith15mlofTBS/T. temperature. markers in10mlofblockingbufferwithgentleagitationfor1houratroom HRP-conjugated anti-biotinantibody(1:1000)todetectbiotinylatedprotein antibody(1:2000)and Incubate membranewithHRP-conjugatedsecondary Wash threetimesfor5minuteseachwith15mlofTBS/T. antibodydilutionbufferwithgentleagitation primary antibody(attheappropriatedilution)in10ml Incubate membraneandprimary Wash threetimesfor5minuteseachwith15mlofTBS/T. Incubate membranein25mlofblockingbufferfor1houratroomtemperature. minutes atroomtemperature. (Optional) Aftertransfer, washnitrocellulosemembranewith25mlTBSfor5 proper exposuretime. wrap andexposetox-rayfilm.Aninitial10-secondexposureshouldindicatethe wrapinplastic Drain membraneofexcessdevelopingsolution(donotletdry), ® substratecanbefurtherdilutedifsignalresponseistoofast. ® incubationanddeclinesoverthefollowing2hours. 2 ) ofmembrane;fordifferentsizedmem Web ® (0.5ml20XLumiGLO n www.cellsignal.com overnight at4°C. ® , 0.5ml20X -

page  of 2