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`SAAD AAGHVA, MLE, MYS APIS Jas, $Y Fe Mase SYPsaeejso| 24
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`esa AU, MS ATs dogs Sept ees YABY EE Gereada) sep 7s]
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`
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`(phosphorylase), #4 AX
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`
`Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg | Ala-Ser (AS 1H
`
`dole},
`
`& AAloaol4y, GTILIMWA) 8&3 BYAS AAA. GSTILIUWA) 6y BYaeSe als SEWA3 =}
`AL(GST), 91Zb IL-11 # o]S Apolo] AUS BHA
`coe TARA. GST-ILI(Ae] ESA ap EeQE
`GST-ILIUAS AZ#S7] Ma, o]. Sebo] Ba BSAAqeS DGEXAT-BILTLCA) AZASH(E 1). Ws
`
`ILIADS GSSSE DNA ASS OZ IL-11 cDNARE] PCR @ APO E-G aElE FEA 2 (site-directed
`mutagenesis) Jah +S 3-9t+.
`PCR] ARES S}7] Szho]4 BL 37] S+ Set:
`
`5': GGA TCC CCG CGA GCT TCC CCA GAC CCT (4S He: 12)
`
`
`
`(AAS HS: 16) AS HSeaSages DV. BamHl/Sall Azle
`PCR -& 5' “#bo) 4 = Pro-Arg-Ala-Ser
`DNA #Z}S pGRX4T-1 SE}AE] ES] Bani /Sall BIS SEYAA pCETHILUAS ALAA. Sepanpe
`pGEMT-12 Ae GST Aa Yo] SEY AA 29) Leu-Val-Pro-Arg-Gly-Ser (SEQ ED NO:1)& 2h. Ope},
`pGEX4T-hIL11(A)= BREE S S-EWMAyt AS O1Zb I-11 4S Apolo] WE? ESA AG #4) Lev-Val-
`Pro-Arg-Gly-Ser-Pro-Arg | Ala-Ser (A7@o HE: 8)S BET.
`
`OFAWS] TL-11(NCBI PEF HS: AAAS9132)9°] Asp155S Asn& st HZ] Sa] Abo] E-Aee Fepay4Aas +
`Baap.
`
`B#n 4d 9S SASS ILIA) ofF] 4A ASS Sy] Gea:
`
`Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg | -Ala-Ser-Pro-Asp-Pro-Arg-Ala-Gli-Leu-Asp-Ser-Thr-Val-Leu-Leu-Thr-Arg-
`Ser-Leu-Leu-Al a-Asp-Thr-Ar g-G1n-Leu-Al a-Al a-G1 n-Leu-Arg-Asp-Lys—Phe-Pro-A! a-Asp-Gly-Asp-His-Asn-Leu-
`Asp-Ser-Leu-Pr o-Thr-Leu-Al a-Met-Ser-Al a-Gl y-Ala-Leu-Gl y-Al a-Leu-G] n-Leu-Pr o-G1 y-Va 1-Leu-Thr-Ar g-Leu-
`
`Arg-Ala-Asp-Leu-Leu-Ser-Tyr-Leu-Ar g-His-Val-G1n-Tr p-Leu-Ar g-Arg-Ala-G1y-G]l y-Ser-Ser-Leu-Lys-Thr-Leu-
`Glu-Pro-Glu-Leu-Gl y-Thr-Leu-G1n-Al a-Arg-Leu-Asp-Ar g-Leu-Leu-Arg-Ar g-Leu-G1 n-Leu-Leu-Met -Ser-Ar g-Leu-
`
`Ala-Leu-Pro-G1n-Pro-Pro-Pro-Asn-Pro-Pro-Al a-Pro-Pro-Leu-Al a-Pro-Pro-Ser-Ser-Al a-Tr p-Gly-Gly-I ]e-Arg-
`
`- 18-
`
`
`
`3': GIC GAC CCC TTA TCA CAG CCG AGT CTT CAG (A7@ We: 13)
`
`BamH]
`
`Sall
`
`a7] Sebolo} BS Apo] E-c] MES HEpaly|A] Aa] AB:
`
`5'DN: OCA GCC ACC COC GAA CCC GCC GGC GCC (AS We: 14)
`
`3'DN: GGC GCC GGC GGG TIC GGG GGT GGC TGG (414 HS: 15)
`
`
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`<90>
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`Arg-Leu (ATS BIS: 17)
`
`BIS 10-2008-0002845
`
`te Seen] = pGEX4T-hILIW(A)S 9]. 2r}°] BL21e YaAslAd. Qadsats gy au(At SFE Oy
`/ml)°]) @Ee LE BHA(Broth4, SE24]7]2, OD 600°] 0.59) BSS BAZ] 87CoA TASH.
`ejoA,, YAMEBR-B-D-BHestttegay =(isopropy|~_G-D-thiogalactopyranoside, IPOS AS SE
`
`0.4 mM] 47s] WMA aS HRSAABS 47H 34/7 Set B7ed4 Vaart. GSI-I_Luse
`Was SDS-PAGESa Selaeau. ASS Agee] ola +Ssw, pH 8.02] 500M BelZ-HC] eso]
`ABHZS RYE PaYAG. ERA PMS A}Ss44 pd 80S FAAGes SAEs
`S SE 5.6n4] AtaeI. CHE Ssolee] a7404 Fagard. SH AWaSs aoe 4
`> k
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`&, ated 22 das 150 oM NaCl @ 10 oe]
`
`NS pH 8.02] 25 mM EF|A-HC] SSMOH aH] MQYs2)71 SEES ABBA
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`Apo] AA) 2) alee 2 HaALBA]ae. Ara
`
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`AYES 449] WSOSE UR AAS © lowe, 304, LAA, 2Vea SALE] ALA Shab Oat -eh
`
`AWS SDS-PAGE] Fa BASU Hep] we Bal
`Hseqeeny A-PagsAA wss S27)gc.
`(Coomassie brilliant blujJ& Be AIso4 7A SA AU. Seb GHA GSThHILMCAS so] AYR] aye}
`Aae 27S] # AVS] GST BB (26 kDa)
`BW IL-11018.2 Wa) 2 2UDAE 2a BV 2) IL-11 Hoye) 4
`GD AGS B27) Sate. GT Be 4A NE Su HA PACA 2B Ut Be= Hoya a
`B9°2 AABMH(E 2a BR 2b).
`
`&o{mt ej
`HS ALSAA] ojos, IL-11lS SHES AeA A VS HES Asbo A-Ber}Sy spo}
`Se DASAOT YES AAA.
`IL-1le] WEE fat YAS MeApo|s 191A HT GMA AaZ
`
`(Procise 491A HT protein sequencer, tho]= Hho] 2 ATAZBMA (Applied Biosystems), "|)a)S ARs HB
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`(AAS HS: 18) VAS AVIS Bolsa.
`
`oo] CF lomg/ml9] SHEE Se AeA 4 VS FE Was GaSeAge 4
`STHie YARUN. ABS SHES Wyzs24 us $F a= WAsu
`
`rots pH 8.02] sec} Be] cf ome] AA PMWM BSS oF Vr. UHlejo} Be
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`
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`
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`
`IL-1L 4A, Ps WE Ato] SUAS Sa] Ha, IL-LS Sole wae ASE DAWE S7zZ
`Aale = f°)2 AEM EIAWAS ayaa, B-E=/MS(MALDI-TOR/MS) (222a SFA(Proteomics
`Solution)
`I(#°F4|(Voyager)-DE STR), GSrbo]= wo] Sa AMA)Bol #ASAc. AE (Compute)
`pl/MW 4] (au.expasy.org/tools/pi_tool .btmloA) 3}4-7}-s S)ol w= IL-112] oat 42-2 18.25 kDae] a
`©}, 18264 Da YA7} PASM Ee 3). AA A4ePe, SE IL-1) AYA] GILUCAY Est 4
`Wee] BIAS Ae) Selspgict.
`
`Us pARoB, 23-Y ABR AYWS He Heys Ae IL-l] PYAS OM IBA US He Was
`ASSO PA AQasasgae. Was Wag Agel Aer pS Aes] BSS oO} HQ.
`IL-11 Yas =
`
`Tree WES eed BS ASSO Of AMS SAA FIT ae] BAA. 24S MWA] Ha, Mes B
`3 2A 71/7 cll 0.15 M Gly-NaQHS SS pH 9.52] SEN ES ASA at. BH Gags 0.15 V
`Gly-NaOH 3 0.15 M NaCl @-F-3H= pH 9.52] MB FS AA Rso] BSA Zc. Ab] Fale] BE BAe
`ACAHAL FBSA.
`
`o]A], Co] 2 3 AVeweE See) AAIL-1] BAAS Q Ayer DS FE yas Asay 47}
`
`= AA. Av] dae Fae AVS 1M GlyNaOdHS SHE ph 9.529] SEN GS APRs Mas o}
`At}. 47] AGS 40 mM Gly-NaQHS @+-s+= pH 9.59] Sed HS ARs ABMAS ol ¥$9q.
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`
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`
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`
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`
`PASM. 40mMGly-NaOHS SHPSH= pH 9.52] AF HS Aol ASA 7] DHS
`7] -S et] pH Ae OF 95a. 4] Bele De Samet] SE Ate
`jet ate Sa qer7} 89228 AARC.
`
`GST-IL11(A) #3 PAYAL MBS BSR Sa 482 Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-AlaSer (JS HS:
`8)S BRAG. AS Leu-Val-Pro-Are-Gly-Ser-Pro-Are-Ala-Ser (97S He: 8)°o]) Se S2)4 4S Lew-Val-
`Pro-Are-Gly-Ser (JS HS: DS Bee], BHAGS Argel Gly7t okArg! Alat7>y Aoyt. ose
`EHS] Leu-Val-bro-Are-Gly-Ser
`(41S WS: 1) U4 Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-Ala-Ser (A]2 WS:
`8s tl ASSAYS Bose}.
`
`<li4>
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`
`ALA] al] 2
`
`GST-E] Al B4
`
`peexar-1-Ranal E] Al B42} CDNAS Asbo] GSE]B4 WA SebAznjeel pGE-e]ezl pas axag
`Te A). SAA SQAAz AA BWS(RI-PCR,
`reverse transcription-polymerase chain reaction)] 9 a
`K562 A] 243 ALBS RABE] CZF ESA PAS SSSISGE DNAS FHYVSIAWH. PCR] AAS
`
`FBaFSsAqe4 ee B7)% Pu:
`
`5':GGATCC CCT OCGA GCT TCT GAC AAA CCC GAT ATG CIS WE: 18)
`
`BamHI
`
`3':GTCGAC TTA CGA TTC GCC TGC TTG CTT CTC (AS HS: 19)
`
`Sal]
`
`5’ Spo] PCR APSO) pGEX4T-1 BA SABAYES SHS 4 AG Leu-Val-Pro-Arg-Gly-Ser-Pro-Arg-
`
`AlaSer (AAS HE: 8)°e] WAYS | BES Gly-Ser-Pro-Arg 1 Ala-Ser (4S HS: 209 tS AAS GHG
`EE aes] Qey.
`
`SSFsl YES pGEM-T o]4| (Easy) SAY E(SZBq
`135 bp cDNA #822 PCR Sa] AAW.
`(Promega), US AASV) SEAS pews BAS AMSA. AS Sel =, pGlw-e]mBas]
`BamHI/Sall 422,
`pGEX4T-12 54] Guaa des Fates Aol” Us a4 2)Bs->> AAA
`pGEX4T-1-Kan Eo] “See SAE SEdagacy.
`
`Bax 2d $9E SMe YA BA ola ASS sls} Se:
`
`Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-
`Leu-Val -Pro-Arg-Gly-Ser-Pro-Arg | -Ala-Ser-Asp-Lys-Pro-Asp-Met-Ala-
`Lys-Leu-Lys-Lys-Thr-Glu-Thr-Ghi-Glu-
` Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Tl ir-I le-Glu-Gm-Glu-Lys-G1n-Ala-
`
`Gly- Glu-Ser (AS HWS: 21)
`
`ae Wels o].Sz}o] Diba kB PaAtsrlaAud. PaAaaTS lB BSBAWA BFAl7/Z OD 6009] 0.54 UE
`
`a a7) 37C A] WaT, IPTGS 0.4 oe] AS SEO] Artaad. ee 3A) 2 Sek 2|SSpar ABS
`AAtezo] Ja] -Ssacy. GST-E|SAl B49] BAS SDS-PAGESal Selaacy. wWeElelo} HasPl 8.0
`
`he] 50 mM E2]A-HC] SEHAaa VT AST Sao] Ja HaAch. GSI-Egs4) p48 2a
`YY AAOeTE SHES ABA 4 DS F-Sol] Sal Balsa.
`
`AAla BE Bas SHOE Psa 15% BelAl He] ALSAbSso}] SMSPaces] Sal BAaArcp.
`
`1A RBA) 2b) eoAA Sha Ste
`
`41°] GST-E] SLA] B4(31 kDa)7} AA ALEEA OF], ELSAl B4(5 kDa) ©] OFS BES al zro] zo] ue} Sr7pa-q
`TH 5)
`
`Este 4+2a 2a] ol44, A B4E =an ASEA 4 1S HSS ASSO AAEMEay
`Ho] SH AAAS GT FES AAAAr. EMA BAe} NW Ofer AAS MEAPO|S 491A HT BHA
`AA(FSS Ho] QaA aga, ways n}-G-8h0 eAaar, wes YEA B47ye 4 N-Beo] AS
`
`
`
`-?0-
`
`<123>
`
`<126>
`
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`
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`
`pes] AHA BSS 20Co}A 150 mM NaCl
`
`
`
`1 S24 BSH 0.5 Feel Ja Agead.|
`
`¢
`
`100
`
`B@ BAe exe]2 10 mM warere a 8,09| 25 mM2] vale
`SU WH SHLEW S-HVA IAB 2 BAA.. etsay
`
`
`
`<129>
`
`<130>
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`<13l>
`
`<132>
`
`Ala-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-lle (44
`
`We:
`
`BIS 10-2008-0002845
`
`Al Baro] Go] o] up
`
`aefaau)
`“Be|* SSB2qyS S72 Aaa, 8e]-
`HES]ees Ya eAaaty.
`
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`J HS EA B42] aed WARS 5.02 Kalle, 4992 Da Fa7t BAHUHCE 6). cle] Aye
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`cp FHS4 64 AYA¢) GSES 649 ERY AAS] AGES Ao] HVS ar.
`
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`°] tte]
`
`Nau,expasy.org/tools/pi_toc]html] Qeahs
`
`Ala 3
`
`GST-IL6
`
`Meso] GST-IL6 BA BepAcjeY
`cDNAS 4
`IL-69]
`4]
`RT-PCR]
`pGEX(T-1°]]
`pGEX-IL6S A)AatBrC 7).
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`AA pzPAlBEE) AAS HS RNABALE] F IL-6S HSBSE DAS
`Zedaacd. PCR) Are
`@ Sy wyPSIQe ee a7]9 Aq:
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`<133>
`5'!GGATC

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