Modification of Superoxide Dismutase 1 (SOD1) Properties by a GFP Tag – Implications for Research into Amyotrophic Lateral Sclerosis (ALS) 英文参考文献.docVIP

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Modification of Superoxide Dismutase 1 (SOD1) Properties by a GFP Tag – Implications for Research into Amyotrophic Lateral Sclerosis (ALS) 英文参考文献.doc

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Modification of Superoxide Dismutase 1 (SOD1) Properties by a GFP Tag – Implications for Research into Amyotrophic Lateral Sclerosis (ALS) 英文参考文献

ModificationofSuperoxideDismutase1(SOD1) PropertiesbyaGFPTag–ImplicationsforResearchinto AmyotrophicLateralSclerosis(ALS) JamesC.Stevens1.,RuthChia1.,WilliamT.Hendriks2,VirginieBros-Facer3,JanvanMinnen4,JoanneE. Martin5,GrahamS.Jackson6,LindaGreensmith3,GiampietroSchiavo7,ElizabethM.C.Fisher1* 1DepartmentofNeurodegenerativeDisease,UCLInstituteofNeurology,London,UnitedKingdom,2CenterforRegenerativeMedicine,MassachusettsGeneralHospital, HarvardMedicalSchool,Boston,Massachusetts,UnitedStatesofAmerica,3SobellDepartmentofMotorScienceandMovementDisorders,UCLInstituteofNeurology, London,UnitedKingdom,4DepartmentofCellBiologyandAnatomy,HotchkissBrainInstitute,UniversityofCalgary,Calgary,Canada,5ICMSPathologyGroup,Queen MaryUniversityofLondon,London,UnitedKingdom,6MRCPrionUnit,UCLInstituteofNeurology,London,UnitedKingdom,7MolecularNeuropathobiologyLaboratory, CancerResearchUK,London,UnitedKingdom Abstract Background:SincethediscoverythatmutationsintheenzymeSOD1arecausativeinhumanamyotrophiclateralsclerosis (ALS),manystrategieshavebeenemployedtoelucidatethetoxicpropertiesofthisubiquitouslyexpressedmutantprotein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins,andwhethertheybehavesimilarlytotheiruntaggedSOD1counterparts. Methodology/Principal Findings: Here we compare the physicochemical properties of SOD1 and the effects of GFP- tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showedidenticalpatternsofcytoplasmicexpressionandofmovementwithintheaxon.However,GFPtaggingofSOD1was foundtoaltersomeoftheintrinsicpropertiesofSOD1,includingstabilityandspecificactivity.Evaluationofwildtypeand mutantSOD1,taggedateithe