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Biochemical analyses and molecular modeling explain the

functional loss of 17β-hydroxysteroid dehydrogenase 3 mutant

G133R in three Tunisian patients with 46, XY Disorders of Sex

Development

Roger T. Engeli*, Bochra Ben Rhouma*, Christoph P. Sager, Maria Tsachaki, Julia Birk, Faiza Fakhfakh, Leila Keskes, Neila Belguith, Alex Odermatt

Published manuscript

Contribution: Performed in vitro activity assays (Figure 2), western blot (Figure 3),

immunolocalization experiments (Figure 4), and gene alignments (Figure 5). Co-wrote the paper manuscript. First authorship was shared equally (*).

Aims: Biochemical analyses and explanation of the functional loss of 17β-HSD3 in Tunisian

patients with G133R and C206X mutations causing 46, XY DSD.

Results: G133R and C206X mutations lead to a complete loss of 17β-HSD3 function despite

normal protein expression levels.

Conclusion: Mutated arginine residue G133R, causes steric hindrance of the cofactor NADPH

binding. The truncated C206X mutation causes inactivates 17β-HSD3 due to the loss of a specific section of the substrate binding site.

Biochemical

analyses

and

molecular

modeling

explain

the

functional

loss

of

17b-hydroxysteroid

dehydrogenase

3

mutant

G133R

in

three

Tunisian

patients

with

46,

XY

Disorders

of

Sex

Development

RogerT.Engelia,1,BochraBenRhoumab,1,ChristophP.Sagerc,MariaTsachakia,Julia Birka, FaizaFakhfakhb,LeilaKeskesb,NeilaBelguithb,d,**,Alex Odermatta,*

a

DivisionofMolecularandSystemsToxicology,DepartmentofPharmaceuticalSciences,Pharmacenter,UniversityofBasel,Basel,Switzerland

b

HumanMolecularGeneticsLaboratory,FacultyofMedicine,UniversityofSfax,Sfax,Tunisia

c

MolecularModeling,DepartmentofPharmaceuticalSciences,Pharmacenter,UniversityofBasel,Basel,Switzerland

d

DepartmentofMedicalGenetics,HediChakerHospital,Sfax,Tunisia

ARTICLE INFO

Articlehistory:

Received24June2015

Receivedinrevisedform21October2015

Accepted29October2015

Availableonline3November2015

Keywords: 46,XYDSD 17b-hydroxysteroiddehydrogenase HSD17B3 Testosterone Mutation

Structurefunctionrelationship

Malesexualdevelopment

ABSTRACT

MutationsintheHSD17B3gene resultingin 17b-hydroxysteroiddehydrogenasetype3(17b-HSD3) deficiency cause46, XY Disorders of Sex Development (46, XY DSD). Approximately 40 different mutations in HSD17B3 have been reported; onlyfew mutant enzymeshave been mechanistically investigated.Here,wereportnovelcompoundheterozygousmutationsinHSD17B3,composedofthe nonsensemutationC206XandthemissensemutationG133R,inthreeTunisianpatientsfromtwonon- consanguineousfamilies.MutantsC206XandG133Rwereconstructedbysite-directedmutagenesisand expressedinHEK-293cells.ThetruncatedC206Xenzyme,lackingpartofthesubstratebindingpocket, wasmoderatelyexpressedandcompletelylostitsenzymaticactivity.Wild-type17b-HSD3andmutant G133Rshowed comparable expressionlevelsand intracellularlocalization. The conversion of D4- androstene-3,17-dione(androstenedione)totestosteronewasalmostcompletelyabolishedformutant G133Rcomparedwithwild-type17b-HSD3.Toobtainfurthermechanisticinsight,G133wasmutatedto alanine, phenylalanineandglutamine.G133QandG133Fwerealmostcompletelyinactive,whereas G133Adisplayedabout70%ofwild-typeactivity.SequenceanalysisrevealedthatG133on17b-HSD3is locatedinamotifhighlyconservedin17b-HSDsandothershort-chaindehydrogenase/reductase(SDR) enzymes.Ahomologymodelof17b-HSD3predictedthatarginineoranyotherbulkyresidueatposition 133 causes steric hindrance of cofactor NADPH binding, whereas substrate binding seems to be unaffected.TheresultsindicateanessentialroleofG133inthearrangementofthecofactorbinding pocket,thusexplainingtheloss-of-functionof17b-HSD3mutantG133Rinthepatientsinvestigated.

ã2015ElsevierLtd.Allrightsreserved.

1.Introduction

17b-hydroxysteroid dehydrogenasetype 3 (17b-HSD3) defi- ciencyisarareautosomalrecessivecauseof46,XYDisordersofSex Development(46,XYDSD)describedin1971[1].Itiscausedby mutations in the HSD17B3 gene (9q22) encoding the 17b-

HSD3 enzymeconsistingof 310amino acids[2,3].17b-HSD3 is predominantly expressedin thetestesand utilizesNADPHasa cofactor [2,4]. 17b-HSD3 catalyzes the conversion of the D4- androstene-3,17-dione(androstenedione)totestosterone,whichis responsibleforthenormalfetaldevelopmentofmalegenitalia[5]. 17b-HSD3deficiencyischaracterizedbyaspectrumofclinical phenotypes due to a complete loss or residual activity of the mutated17b-HSD3enzymeinthetestes[6],aswellasdifferences inthedegreeofend-organresponsivenessandtimingofexposure ofexternalgenitaliatoandrogens.Theonsetoftheextra-testicular conversionofandrostenedionetotestosteroneby17b-HSD5(also knownasAKR1C3)isresponsiblefortheobservedvirilizationat puberty [1,7–9].The characteristic phenotype atbirth is an XY individualwithundervirilizationoftheexternalgenitalia,which oftenappearfemalewithorwithoutclitoromegalyand/orlabial

* Corresponding author at: Division of Molecular and Systems Toxicology,

Department of Pharmaceutical Sciences, Pharmacenter, University of Basel,

Klingelbergstrasse50,4056Basel,Switzerland.

** Correspondingauthorat:HumanMolecularGeneticsLaboratory,Facultyof

Medicine,MagidaBoulilaStreet,3029Sfax,Tunisia.

E-mailaddresses:[email protected](N.Belguith),

[email protected](A.Odermatt).

1

Theseauthorscontributedequallytothepresentstudy.

http://dx.doi.org/10.1016/j.jsbmb.2015.10.023

0960-0760/ã2015ElsevierLtd.Allrightsreserved.

ContentslistsavailableatScienceDirect

Journal

of

Steroid

Biochemistry

&

Molecular

Biology

fusion and a blind-ending vagina [1,10].Affected patientshave testes and often have normal Wolffian duct derivatives. The diagnosisof17b-HSD3deficiencyisbasedonanincreasedratioof androstenedionetotestosterone.It can besuspectedin case of inguinalherniaorsexualambiguityatearlychildhoodandincase ofseverevirilizationandprimaryamenorrheaatpubertyage[11]. Here, we report on three Tunisian patients with 17b- HSD3deficiencyfromtwodifferentnon-consanguineousTunisian families. Our results revealed novel compound heterozygous mutations,i.e.,theprematurestopcodonC206Xandthemissense mutation G133R, in the HSD17B3 gene responsible for 17b- HSD3 de_ficiency. The impact of the missense mutations was studiedbysite-directedmutagenesis,expressionoftherecombi- nantproteinsinHEK-293cellsandbiochemicalanalysisofenzyme activity. In order to understand the loss of enzyme activity of mutantG133R,additionalsubstitutionsofG133wereinvestigated anda17b-HSD3homologymodelwasgeneratedusingModeller Version9.11[12–14].

2.Experimentalprocedures 2.1.Subjectsandclinicalhistory

ThreeTunisianpatientsdiagnosedwith46,XYDisordersofSex Development(DSD)werestudied.Since birth,allpatientswere raised as girls; patient P1 consulted at the age of 7 years for inguinalhernia,andthetworemainingpatientsweresistersand consultedatpubertyageforprimaryamenorrhea.Forallpatients, the physical examination revealed normal female external genitalia,and thekaryotypeanalysis, performedusingstandard G-banding technique,revealed 46, XY.The magnetic resonance imagingofthepelvisandtheabdomenshowednovisualizationof theuterusorvaginabut revealedinguinaltestes. Theresultsof hormonalbaselinetestingofallpatientsarepresentedinTable1. 2.2.SequencingtheHSD17B3gene

Peripheralbloodsamplesofthepatients(P1,P2andP3)andthe parentsofP1werecollectedandgenomicDNAwasextractedusing phenol-chloroform standard procedures [15]. All exons and flanking intron regions of the HSD17B3 gene were tested for mutations by sequence analysis using the previously reported primers [16]. The PCR was performed using a thermal cycler (GenAmpPCRSystem9700,AppliedBiosystem,Waltham,MA)ina finalvolumeof50mLcontaining50nggenomicDNA,0.2mMof eachprimer,1PCRbuffer,1.2mMMgCl2,0.5mMdNTP,and1U TaqDNApolymerase(PromegaGoTaqDNAPolymerase,Fitchburg, WI).DirectsequencingofPCRproductswasperformedusingthe ABIPrismBigDyeTerminatorCycleSequencingReadyReactionKit (ABIPRISM/Biosystems)andtheproductswereresolvedonanABI PRISM.

2.3.Site-directedmutagenesisandconstructionofexpression plasmids

ApCMV6expressionvectorcontainingthefull-lengthhuman 17b-HSD3 sequence was a kindgift of thelate Prof.Dr. Stefan Andersson.ThecDNAsequencefromtheATGinitiationcodonto thestopcodon,whichwasreplacedbyaFLAGepitopefollowedby a stop codon, was amplified by PCR and inserted into the pcDNA3.0 expression vector (Thermo Scientific, Rockford, IL, USA)betweentherestrictionsitesBamH1andXba1(Roche,Basel, Switzerland).Thisconstructwasusedasatemplatetointroduce the FLAG-tagged point mutations. Point mutations were intro- ducedbysite-directedmutagenesisusingPfuPolymerase(Prom- ega,Madison,WI,USA)(foroligonucleotideprimersequencessee Supportinginformation).Allexpressionplasmidswereverifiedby sequencing.

2.4.Cellcultureandenzymeactivityassay

HumanEmbryonicKidney-293cells(HEK-293,ATCC,Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM,Sigma–Aldrich,St.Louis,MO,USA) supplementedwith 10%fetalbovineserum(FBS,Connectorate,Dietikon,Switzerland), 100U/mL penicillin,100mg/mLstreptomycin (Life Technologies, Grand Island, NY, USA), 10mM HEPES buffer pH 7.4 (Life Technologies,GrandIsland,NY,USA),and1%MEMnon-essential aminoacidsolution(Sigma–Aldrich).Cellswerecultivatedunder standardconditions(37C,5%CO2)inanincubator(ThermoFisher Scientific,Waltham, MA, USA). HEK-293 cells (1.510 6_{) were} seededinto10cmdishes,followedbytransienttransfectionbythe calciumphosphateprecipitationmethodwith8mgofexpression plasmidforwild-type17b-HSD3[17,18]ormutant17b-HSD3(see below).TransfectedHEK-293cellswereincubatedfor24hat37C, and15,000cellswereseededin100mLmediumin96-wellplates, pre-coated withpoly-L-lysine (Sigma–Aldrich). Afterincubation for another24h, themedium was replaced by50mLcharcoal- treatedDMEMandthe17b-HSD3enzymeactivitymeasurements wereperformedbyaddingandrostenedione(Sigma–Aldrich)ata final concentration of 200nM, containing 50nCi [1,2,6,7-3_H]- androstenedione(AmericanRadiolabeledChemicals,St.Louis,MO, USA). After 30min reactions were stopped and cells lysed by adding20mLofmethanolcontaining2mMunlabeledandrostene- dioneand2mMtestosterone(Sigma–Aldrich).Anamountof20mL oflysatewasloadedontoTLCplates(Macherey-Nagel,Oensingen, Switzerland)andsteroidswereseparatedusingchloroform/ethyl acetate at a 4:1 ratio. Corresponding substrate and product concentrations were determined after scintillation counting (PerkinElmer,MA,USA).

2.5.Immunostaining

HEK-293cellswereseededonglasscoverslipsandtransiently transfectedwithplasmidsforFLAG-taggedwild-typeandmutant 17b-HSD3 constructs after 24h. At 48hpost-transfection cells werewashedwithPBS,fixedwith4%paraformaldehydeandcell membraneswerepermeabilizedfor5minwith0.3%TritonX-100. Afterblockingwith2%defattedmilkinPBSfor30min,cellswere incubatedwiththeprimaryantibodyatadilutionof1:100for1h at room temperature. Rabbit anti-FLAG (Sigma–Aldrich) and mouseanti-proteindisulfideisomerase(PDI)antibodies(Abcam, Camdridge, UK) were used. Anti-PDI antibodies were used as controlforaproteinwithanendoplasmicreticulumdistribution. Afterwashing,cellswereincubatedfor30minatroomtempera- turewithgoatanti-rabbitAlexaFluor1488andgoatanti-mouse AlexaFluor1555IgG(Sigma–Aldrich)atadilutionof1:300.After washing,samplesweremountedinMowiol4-88(Roth,Karlsruhe,

Table1

Characterizationofpatients.

Patient1(P1) Patient2(P2) Patient3(P3)

Age(years) 7 14 15 Height(cm) 155 – – Weight(kg) 45 – – Tannerstage P1B1 P4B1 P5B1 Testosterone(ng/mL) 0.8 3 4 LH(IU/L) 12.5 – – FSH(IU/L) 4.7 40 42 Caryotypeanalysis 46,XY 46,XY 46,XY

Referencesvalues:Luteinizinghormone(LH):1.24–8.62mUI/mL;folliclestimulat-

Germany)and analyzedusing anOlympus Fluoview1000laser scanningconfocalmicroscope(Olympus,Tokyo,Japan).

2.6.Westernblot

HEK-293cells(300,000)wereseededin60mmdishes.After24h, thecalciumphosphatetransfectionmethodwasusedtotransfect 5mgofplasmidforFLAG-taggedwild-typeandmutantenzymes. Mediumwaschangedafter4handcellswereincubatedforanother 48h.CellswerelysedusingRIPAbuffer(Sigma–Aldrich),containing proteaseinhibitorcocktail(Roche,Basel,Switzerland),andcentri- fugedat14,000_gfor20minat4_{C.Thesupernatantwascollected} and protein concentrations were quantified using the Pierce1 biocinchonicacidproteinassaykit(ThermoScientific,Rockford,IL, USA).SampleswerepreparedinLaemmlisolubilizationbuffer(LSB) (5mMTris–HCl,10%glycerol,0.2%sodiumdodecylsulfate,0.04% bromophenol blue, pH 6.8), containing 5% b-mercaptoethanol (Promega,Madison,WI,USA)andboiledfor5min.Anamountof 35mgoftotalproteinwasseparatedbysodiumdodecylsulfate- polyacrylamidegelelectrophoresis(SDS-PAGE)ona12%acrylam- idegelandtransferredtoImmun-Blot1polyvinylidenedifluoride (PVDF)membranes(Bio-RadLaboratories,Hercules,CA,USA).For detectionoftheFLAGepitope,themembranewasblockedusing3% defattedmilkin PBSfor 30minand incubatedwiththemouse monoclonalM2antibody(Sigma–Aldrich)atadilutionof1:750in blocking solution overnight at 4C. After washing with Tris- bufferedsaline(20mMTrisbuffer,pH7.4,140mMNaCl)containing 0.1%Tween-20(TBS-T),themembranewassubsequentlyincubated withhorseradishperoxidase-conjugatedgoatanti-mousesecond- aryantibody(Sigma–Aldrich)for1hatroomtemperature.Forthe detectionofthehousekeepingcontrolcyclophilinA,blockingwas performed overnight at 4C using 3% defatted milk in PBS. Subsequently,themembranewasincubatedwiththerabbitanti- humancyclophilinApolyclonalantibody(Abcam,Camdridge,UK) at a dilution 1:2000 in blocking solution for 1h at room

temperature. After washing with TBS-T, the membrane was subsequentlyincubatedwithhorseradishperoxidase-conjugated goatanti-rabbitsecondaryantibody(Sigma–Aldrich)atadilution 1:2000in3%defattedmilkinPBS.Afterwashing,theproteinbands were visualized on a Fujifilm ImageQuantTM LAS-4000 (GE Healthcare,Glattbrugg,Switzerland)usingtheImmobilonWestern ChemiluminescentHRPsubstratekit(Merck,Kenilworth,NJ,USA). ThebandsobtainedfortheFLAG-taggedwild-typeandmutant17b- HSD3protein weresubjectedtodensitometry analysisusingImageJ software.SignalswerenormalizedtothoseofcyclophilinAhouse keepingcontroltocorrectforloadingdifferences.

2.7.Molecularmodeling

Protein sequenceswerealignedbymultiplealignment using the MultipleSequence Viewerimplemented in Maestro[19]. A homologymodelofwild-typeaswellasoftheG133Rmutantof 17b-HSD3 were obtained by using the aligned sequences (see Supportinginformation)togetherwithacrystalstructureof17b- hydroxysteroid dehydrogenase 1 (17b-HSD1, PDB code: 3DHE, 2.3Å)asinputforModeller(Version9.11),providedbytheMPI BioinformaticsToolkit[12–14].

SiteMapwasusedtopredicttheandrostenedionebindingsite oftheobtainedhomology modelandwassubsequentlyusedas inputforgeneratingaGlideDockingGrid[20–24].TheGlideXP docking protocol was used to dock androstenedione to the generated rigid docking grid. The protein–ligand complex was refined usingPrime [25–27]. Figures wereproduced byPyMOL

[28].

2.8.Multipleproteinalignment

Multiple protein alignment of relative 17b-HSDs was per- formed using ExPASy (http://embnet.vital-it.ch/software/Clus- talW.html).Proteinaminoacidsequencesofalltested17b-HSDs

Fig.1.Mutationalanalysesof46,XYDSDpatients.

AutomatedDNAsequencingoftheHSD17B3geneforthethreepatients:thesequencesrevealedaheterozygoussubstitution(cDNAposition618,C>A)inexon9anda

heterozygoussubstitution(cDNAposition397,G>A)inexon5ofpatientsP1,P2andP3.(A)Resultsofthemutationalanalysisofexon9andexon5forpatientP1andsomeof

herfamilymembers.(B)Resultsofthemutationalanalysisofexon9andexon5forpatientsP2andP3.Symbolsindicatesexphenotype:circlesrepresentfemales,squares

representmales.FilledsymbolsindicatepatientswithmutatedHSD17B3allelesandhalf-filledsymbolsindicateonemutatedallele.Consanguineousmarriageisindicatedby

werecollectedfromthegenedatabasefromtheNationalCenterof BiotechnologyInformation(http://www.ncbi.nlm.nih.gov/gene/). 3.Results

3.1.17b-HSD3mutationG133Roccursin46,XYDSDpatients ThefamilialhistoryofP1recordedapaternalcousinwith17b- HSD3deficiencyduetoahomozygousmutation(cDNAposition 618,C>A)inexon9resultinginthesubstitutionofthecysteineat position206toaprematurestopcodon(C206X)[29].Therefore, thegenomicDNAfrompatientP1wasanalyzedformutationsin theHSD17B3 gene. For patientsP2 and P3, due tothesigns of virilizationobservedattheageof pubertyand theabsenceofa completehormonalprofileincludinganhCGstimulationtest,two deficiencies wereinitially suspected: 5a-reductase2 deficiency with loss of function mutations in the SRD5A2 gene or 17b- HSD3deficiency. DNA analysisof the SRD5A2 gene showed no abnormalitiesintheentirecodingregionandtheadjacentintron/ exonboundaries.Therefore,thegenomicDNAofthepatientswas analyzedformutationsinHSD17B3.PatientsP1,P2andP3were heterozygous for the previously described nonsense mutation C206X. In addition, they were also heterozygous for a novel missensemutation(cDNAposition397,G>A)inexon5,resulting in themutation G133R (Fig.1A and B). The verification of the transmissionofthemutationsamongthefamilyofP1showedthat themutationC206XwascodedbyapaternalalleleandG133Rbya maternalallele(Fig.1A).

3.2.TruncationC206XandsubstitutionG133Rcauseabolished17b- HSD3activity

ThemutationC206X,derivedfromthepaternalallele,causesa truncationoftheenzymeatposition206,onlyfouraminoacids downstreamoftheessentialcatalyticsite(Y198andK202)[30]. The truncatedenzyme lacks a significant partof thesubstrate bindingsite. Enzymeactivitymeasurementswereperformedin intact HEK-293 cells transfected with expression plasmids for eitherwild-typeormutant17b-HSD3enzymes.Theformationof testosteronewasexaminedafterincubationofcellsfor30minwith 200nMandrostenedione.Noactivitycouldbedetectedformutant C206X,evenafterprolongedincubationtime(notshown),andan almostcompletelossofactivitywasobtainedformutantG133R (Fig.2).Sincethesubstitutionofaglycinebyanarginineresidue alters both size and charge of the side chain, three additional mutantenzymeswereconstructed,i.e.,G133A,G133QandG133F. Enzymaticanalysisrevealed that substitution of glycinebythe bulky phenylalanine and by the non-charged glutamine also almostcompletelyabolished17b-HSD3activity,whereasmutant G133Aretainedapproximately70%ofwild-typeactivity(Fig.2).

Next,WesternblottingofFLAG-taggedwild-typeandmutant 17b-HSD3proteinswasconductedtoinvestigatewhethertheloss ofenzymaticactivityofthemutantenzymeswasduetoimpaired proteinexpression(Fig.3).Wild-typeandG133mutantproteins yieldedonespecificbandatabout35kDa,asexpected.Densitom- etry analyses of three independent experiments did not yield significantdifferences intheexpressionlevelsof wild-typeand G133mutantproteins(datanotshown).Aspecificbandformutant C206Xcouldbedetectedatabout18kDa;however,theexpression level seemed to be low and the band was visible only after prolongedexposureoftheblot,whichalsoledtothedetectionof severalunspecific bandsasshown bytheemptyvectorcontrol sample.Furtherinvestigationbyimmunofluorescencestainingand confocalmicroscopyconfirmedthetypicalendoplasmicreticulum (ER) membrane localization of FLAG-tagged wild-type and G133mutant17b-HSD3enzymes(Fig.4).Nosignsofdislocation

ordegradation ofthe G133mutant enzymeswereobserved.In contrast,formutantC206X,despiteitsmoderatesignal,signsof aggregatedproteincouldbedetected,suggestingimpairedfolding. 3.3.G133isconservedamong17b-HSDenzymes

Asequencealignmentofhuman17b-HSD3with17b-HSD3of otherspeciesrevealedthatG133ishighlyconserved(notshown). Moreimportantly,alignmentwithsequencesfromrelated17b-HSD enzymescorrespondingtotheregionoftheturnbetweenb4–a5on 17b-HSD3 showed that the glycine residue is highlyconserved (Fig.5).Allofthe13analyzed17b-HSDenzymesoftheshort-chain dehydrogenase/reductasefamily(17b-HSD1-14,withtheexception of17b-HSD5 thatbelongs to thefamily ofaldo-ketoreductases(AKR) andwasthereforenotincludedinthealignment[31])possessa glycineatthisposition.Theglycineisthelastresidueofaclusterof sevenconservedaminoacidswiththeconsensussequence(I/V)(I/L/ V)(I/V)NN(A/V)G.Theseresiduesareformingtheturnbetweenthe 4thb-sheetandthe5tha-helixoftheconservedSDRstructure.A glycinecorrespondingtoposition133on17b-HSD3isalsofoundin

Fig.2. Enzymaticactivityofwild-typeandmutant17b-HSD3.

HEK-293cells(15,000cellsperwellofa96-wellplate)weretransientlytransfected

withexpressionplasmidsforwild-typehuman17b-HSD3andmutantsG133R,

G133A,G133QandG133F.Enzymaticactivitywasmeasuredbyincubatingcellsfor

30min at37_{Cwithandrostenedioneata finalconcentrationof200nM and}

containing 50nCi [1,2,6,7-3

H]-androstenedione, followed by analysis of the

conversion of androstenedione to testosterone by scintillation counting. The

percentageoftestosteroneformedfromtheinitiallysuppliedandrostenedioneis

shown.ResultsrepresentmeanSDoffourindependentexperiments.

Fig.3. Westernblotofwild-typeandmutant17b-HSD3.

HEK-293cellsweretransientlytransfectedwithplasmidsforC-terminallyFLAG

epitope-taggedwild-typeandmutant17b-HSD3.Afteranincubationtimeof48h,

transfectedcellswereharvestedandequalamountsoftotalproteinwereseparated

bySDS-PAGEandsubjectedtoWesternblottingusingamouseanti-FLAGantibody

fordetection.CyclophilinA(PPIA)wasusedasacontrolandanalyzedusingananti-

Fig.4.Immunolocalizationofwild-typeandmutant17b-HSD3.

In document BOJA. Sumario JUNTA DE ANDALUCIA. 1. Disposiciones generales PÁGINA. Boletín Oficial de la Junta de Andalucía (página 78-81)