V5-CD2AP V5-NEK8. V5-Bicc1. V5-Bicc1 V5-GFP. - V5-Bicc1 - V5-GFP. - Flag-Anks6. Anks6- i
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- Nancy Marshall
- 5 years ago
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![nti-v5 Lysts: nti-flg Flg- - mrna lvls [%] 12 8 4 -i - + Tt - i - + Tt γ-tuulin γ/.tu. γ/.tu. Ho Supplmntry Figur 1.](/docs-images/91/107044019/images/1-2.jpg "Bining o NEK8 to humn n rt orthologus o () Flg tgg ull-lngth rt n humn wr o-trnst with V5 tgg ull-lngth NEK8 in HEK293T lls.")
1 Supplmntry Fig.1 Flg- Flg-ANKS V5-GFP V5-CD2AP V5-Bi1 V5-GFP V5-CD2AP IP: nti-v5, WB: nti-flg V5-Bi1 - Flg V5-Bi1 - - V5-CD2AP V5-GFP IP: nti-v5, WB: nti-v5 Lysts: nti-flg Flg- - mrna lvls [%] i - + Tt - i - + Tt γ-tuulin γ/.tu. γ/.tu. Ho Supplmntry Figur 1. Bining o NEK8 to humn n rt orthologus o () Flg tgg ull-lngth rt n humn wr o-trnst with V5 tgg ull-lngth NEK8 in HEK293T lls. Atr immunopripittion with n nti-v5 ntioy, NEK8 ws prsnt in th pripitts o rt n humn n mous Bi1 whih srv s positiv ontrol. () Quntittiv rl tim polymrs hin rtion (qpcr) or mrna o totl RNA xtrt rom -i lls. Th knokown ws inuil y ttrylin n rs mrna lvls; rror rs: stnr vition. () Wstrn lot o -i ll lysts. protin ws not tt in knokown lls (+Tt). γ-tuulin srv s loing ontrol. () Immunostining o IMCD3 lls or (grn) n γ-tuulin/tylt tuulin (mgnt) (sl ois, r rrowhs; ughtr ntriol yllow rrowhs) onirm loliztion o to th proximl ilium. Hohst (Ho, lu) ws us or nulr stining. Sl rs; 1 µm. Ntur Gntis: oi:1.138/ng.2681
![WT nks6 2 WT nks6 2 Supplmntry Fig.2 nks6 1α µm nks6 1.2 2 H2O H2O 1 - yst ormtion [%] 1 8 6 4 2 355 491 173 121 yst ormtion [%] 1 8 6 4 2 195 26 219 24 3ng 3ng nks6 2 2ng nks6 1 +.](/docs-images/91/107044019/images/2-0.jpg "1ng 3ng 2ng nk8 1ng nks6 1 + 2ng nk8 yst ormtion [%] 1 8 6 4 2 458 4 419 387 yst ormtion [%] 1 8 6 4 2 5 285 335 21 2ng 2ng nk8 2ng nks6 1 1ng nk8 + 1ng nks6 1 2ng 1ng nks6 1 1ng nphp3 1ng nks6 1 +")
2 WT nks6 2 WT nks6 2 Supplmntry Fig.2 nks6 1α µm nks H2O H2O 1 - yst ormtion [%] yst ormtion [%] ng 3ng nks6 2 2ng nks ng 3ng 2ng nk8 1ng nks ng nk8 yst ormtion [%] yst ormtion [%] ng 2ng nk8 2ng nks6 1 1ng nk8 + 1ng nks6 1 2ng 1ng nks6 1 1ng nphp3 1ng nks ng nphp3 Supplmntry Figur 2. Zrish knokown iiny n spiiity () In vitro trnslt zrish protin ws tt in wstrn lot y ntioy. protin lvl rs in th prsn o 2µM o th trnsltion loking nks6 1. () RT-PCR o lysts rom 24hp ol zrish ontrol mryos n nks6 morphnts. Th spli loking nks6 2 trgt th xon1/intron1 ounry. Th RT-PCR prouts show th iiny o th nks6 knokown n squning o th insrt intron prts (rrows) rvl tht th insrtions rsult in prmtur stop oon. Th hous kping gn 1α ws mplii s positiv ontrol. () Quntiition o pronphri yst ormtion us y two inpnnt nks6 s. Injtion o nks6 spli loking 2 us 23% n th trnsltion loking 1 rsult in 31% yst ormtion. nks6 1 ysti phnotyp ws rsu y rt RNA ( p<=.1). () Th omin knokown y qul onntrtions o nks6 1 n nk8 xhiit n inrs pronphri yst ormtion in zrish ( p<=.1); Mnn Whitny rnk sum tst; rror rs: SEM. () Simultnously injtion o hl o th onntrtions us or th singl knokown i not inrs th numr o yst ormtion. () Simultnous injtion o qul onntrtions o nks6 1 n nphp3 us n inrs o yst ormtion ( p<=.1); Mnn Whitny rnk sum tst; rror rs: SEM. Ntur Gntis: oi:1.138/ng.2681
3 Supplmntry Fig.3 ' '' stg 3 nk8 sns ntisns ' stg 37 sns ntisns B' nks6 stg 39 stg 36 stg 31 sns sns ntisns ' ntisns ' sns ntisns stg ODC -RT nk8 nks6-4 ps - 4 ps ps ps Supplmntry Figur 3. Sptil n tmporl xprssion pttrn o nks6 n nk8 in Xnopus (-) Ltrl viws o Xnopus mryos tr WISH or nk8 n nks6 t init t stgs. (-) Ngtiv ontrols with th sns pro. ( - ) Spii stining y th ntisns pro. nk8 n nks6 xprssion ws irst ttl t stg 3-31 (rrows). At stg 36 to 39 nk8 n nks6 ws xprss in th proximl prt o th rnl tuuls (rrows). Sl rs; 5 µm. () Lysts rom mryos o init stgs wr us or RT-PCR. Primr spii or th hous kping gn ODC (ontrol), nk8 n nks6 wr us. Th mpliition o th isolt RNA (-RT) with ODC primrs srv s ngtiv ontrol. nk8 trnsripts wr onstitutivly prsnt twn stg 1 n stg 42 whrs nks6 ws irst xprss t stg 14. Ntur Gntis: oi:1.138/ng.2681
![tuul imtr [mm] Supplmntry Fig.](/docs-images/91/107044019/images/4-0.jpg "4 x µm nk8.")
![kiny lngth [mm].3.2.1 46 17 118 nk8 +.](/docs-images/91/107044019/images/4-4.jpg "1ng nk8 RNA stg35 stg39 stg42 irn kiny")
![lngth [mm].4.2 39 47 47 29 nks6 +.1ng+.](/docs-images/91/107044019/images/4-5.jpg "3ng nk8 RNA i irn kiny lngth [mm].4.")
4 tuul imtr [mm] Supplmntry Fig.4 x µm nk8.2 2 Uniltrl trgt Injtion t th 4-8 ll stg Tissu rstrit xprssion / knokown t stg 39 (tpol) Nk8 E1 E2 I2 E3 I3 E4 I4 E5 I1 E1 E2 E3 E4 I4 E5 E6 stop WT nks6 ODC I5 E6 g stg42 stg39 stg35 nks6 -RT nks6 h injt uninjt nks6 injt uninjt irn kiny lngth [mm] nk8 +.1ng nk8 RNA stg35 stg39 stg42 irn kiny lngth [mm] nks6 +.1ng+.3ng nk8 RNA i irn kiny lngth [mm].4.2 nks stg 35 stg 39 stg j inj uninj nks6 Supplmntry Figur 4. Xnopus morpholino iiny n spiiity () In vitro trnslt Nk8 protin is olish in th prsn o 2μM o nk8. () Shmti illustrtion o nks6 spli loking trgting xon4/intron4 ounry. Th positions o th primrs us or RT-PCR nlysis r init y lu rrows. Squning o RT-PCR prouts illustrt in () rvl tht rmshit ws using prmtur stop oon. E, xon; I, intron. () Spiiity ontrol o nk8. Th rnl phnotyp o nk8 plt morphnts oul rsu y o-injtion o nk8 mrna ( p<=.1); Mnn Whitny rnk sum tst; rror rs: SEM. () Th irn o kiny lngth us y nks6 pltion ( p<=.1) oul not rsu y nk8 mrna; Mnn Whitny rnk sum tst; rror rs: SEM. () Shmti illustrtion o uniltrl trgt injtion o GFP. Th luorsnt signl ws rstrit to on si o th tpol. (g) Th gross morphology o nks6 injt mryos ws not ltr. No vlopmntl ly ws osrv t ny stg. Sl rs; 5 μm. (h) Stining with th pronphri spii ntiois 3G8 n 4A6 ws prorm on Xnopus mryos t init stgs. Shortning o th tuuls o th injt si ws osrv n quntii in (i) Sl rs; 2 μm. (j) Msurmnt o tuul imtr in ltin-stin mryos uniltrlly injt with nks6 tt n nlrgmnt on th injt si (n=47; p<=.1). Ntur Gntis: oi:1.138/ng.2681
5 Supplmntry Fig.5 injt uninjt lhx1 nk8 nk8 nks6 nk8 injt uninjt sl51 nphp3 injt nphp3 uninjt sl44 sl121 irn kiny lngth [mm] ** * nphp3 +.3ng +.5ng NPHP3 tp11 nphp3 injt sl51 uninjt sl44 sl121 PT IT1 IT2 DT1 DT2 sl51 sl44 sl121 tp11 tp11 Supplmntry Figur 5. Chrtriztion o th pronphri phnotyp o nk8 n nphp3 knokown in Xnopus () Biltrlly nk8 n nphp3 injt Xnopus mryos vlop m in ontrst to ontrol mryos. Sl rs; 5µM. () Imgs o ltin stin pronphros o nphp3 morphnts show simplii proximl tuul similr to nk8 n nks6 morphnts. Quntiition o th nphp3 rsu xprimnt y o-injtion o NPHP3 RNA (* p=.11; ** p=.3; p=<.1); t-tst; rror rs: SEM. () Shmti illustrtion o th xprssion o sgmnt spii pronphros mrkrs. Th soium gluos trnsportr sl51 (SGLT-1K) is xlusivly xprss in th proximl tuul n nphrostoms. Th soium iront trnsportr sl44 (NBC1) is xprss in th proximl tuul n rly istl tuul ut not in th intrmit tuul. Th soium/potssium o-trnsportr sl121 (NKCC2) is spiilly xprss in th intrmit sgmnt. Th β1 suunit o th tp11 (N-K-ATPs) is xprss long th ntir lngth o th pronphros. () Uniltrl injtions o nk8 n nks6 t th our ll stg wr ollow y WISH or th trnsription tor lhx1. No irn in th xprssion ws osrv twn th injt n th uninjt si o th mryos t stg 28. Sl rs; 2 µm. () Atr uniltrl injtion o ithr nk8 () or nphp3 () WISH or irnt pronphri sgmnt mrkrs ws prorm. Th xprssion o sl51, sl121 n tp11 ws ru on th injt sis (rrows). Sl rs; 2 µm. Ntur Gntis: oi:1.138/ng.2681
![6 84.4 112.2 14 Position ANKS6 irn kiny lngth [mm].3.2.1 n.s. ** 41 46 31 42 nks6 + +. Q433R Supplmntry Figur 6.](/docs-images/91/107044019/images/6-1.jpg "Humn muttions in ANKS6 () Intiition o rssiv muttions in ANKS6 in six milis with nphronophthisis. Exon strutur o humn ANKS6 DNA (without UTR). Positions o strt oon (ATG) n o stop oon (TGA) r init.")
6 Supplmntry Fig.6 ATG TGA ,616 p ANK 466 ANK ANK ANK ANK ANK ANK ANK ANK SAM 871 A G>C (Al312Pro) B A>G (Gln441Arg) A C>G (3' spli) B _264l (His685Pros*12) A _2372l (Tyr79*) NPH A>C (3' spli) NPH r ousins Fmily: NPH316,Chromosom: Position ANKS6 irn kiny lngth [mm] n.s. ** nks Q433R Supplmntry Figur 6. Humn muttions in ANKS6 () Intiition o rssiv muttions in ANKS6 in six milis with nphronophthisis. Exon strutur o humn ANKS6 DNA (without UTR). Positions o strt oon (ATG) n o stop oon (TGA) r init. Domin strutur o th ANKS6 protin, onsisting o nin nkyrin rpt omins (ANK), n stril lph moti omin (SAM) t th C-trminl n. For th muttions tt rrows init positions in rltion to xons n protin omins. () Livr iopsy spimn rom Ptint B6794 showing mronoulr irrhosis. In th onntiv tissu, svr utulr rtion is sn. Th rtris r pprntly unompni y intrloulr il uts (Piro-Sirius stin highlighting ollgn); sl r,.5 mm. () Nl or iopsy o livr rom ptint A showing ln portl irosis with portl to portl riging irosis. Th ntrl vin is unrmrkl n thr is no signiint hptitis. Vn Gisn stin lt (irosis highlight in r) n hmtoxilin/osin (HE) stin right o uppr pnls; sl rs,.5 mm. Lowr pnls show highr mgniition o th HE stin tissu; sl rs,.1 mm. () Pigr o onsnguinous Turkish mily (NPH316) with nphronophthisis n orti stnosis. Linkg nlysis rvl uniqu homozygous rgion o 8M spnning th ANKS6 lous on hromosom 9. W susquntly intii homozygous muttion o ANKS6 ting th onsnsus ptor spli sit in intron 14 tht sgrgts with th iss. () Vlus o non-prmtri th logrithm o os (LOD) sors ross hromosom 9 o mily NPH316: X-xis givs Aymtri 25k SNP positions (M) n Y-xis givs th (LOD) sor vlus. Th mximl LOD sor o 2,47 ws otin or th homozygous rgion spnning th ANKS6 lous. () Quntiition o Xnopus rsu xprimnts o nks6 with th Q433R mutt rt RNA orrsponing to th ptint muttion Q441R (B7397). Mutnt RNA oul not mliort th rnl nks6 phnotyp. ( p<=.1; ** p=.6; n.s. p=.987); t-tst; rror rs: SEM. Ntur Gntis: oi:1.138/ng.2681
7 Supplmntry Fig.7 A _2372 ITCA p.y79* AAA TAG CCC Lys * A >g Spli sit tgg GT GGC A G>C p.a312p G ATT CCC GAT Il Pro Asp NPH > Spli sit gg GGA CG NPH > Spli sit gg GGA CG NPH > Spli sit gg GGA CG AAA TAT CAG CCC Lys Tyr Gln Pro tg GT GGC ATT GCC GAT Il Al Asp NPH Ht. rrir g g GGA CG NPH Ht. rrir g g GGA CG B A>G p.q441r B _264l p.h685psx V5-Q433R V5- - V IP: nti-flg, WB: nti-v5 - Flg-NPHP3 - Flg-INVS - Flg-NEK IP: nti-flg, WB: nti-flg - Flg-GFP - V5-1 - Lysts: nti-v5 A.Tu -Vnus A.Tu -Vnus Ho IMCD.-Vnus IMCD..Q433R-Vnus Supplmntry Figur 7. Humn ANKS6 muttions () Chromtogrms o six irnt ANKS6 muttions tht wr oun in iniviuls with nphronophthisis. Fmily numr, muttions n prit trnsltionl hngs r init. Squn trs r shown or muttions ov norml ontrols or htrozygous rrirs. Mutt nulotis r init y rrowhs. A lt inits 3-p ltion in wil typ squn o A nots homozygous muttions. () Ultrsoun invstigtion o th kinys in ptint B6794. Th right kiny hs inrs hogniity ompr with surrouning livr prnhym. A mroyst is init y th rrow. Th lt kiny xhiit svrl smll ysts (rrows) in th ortx n th mull. With prmission o Dpt. o Riology, Arhus Univrsity Hospitl, Arhus, Dnmrk. () Co-immunopripittion o V5 tgg rt or th missns mutnt Gln441Arg (Q433R) y Flg tgg INVS, NPHP3 n NEK8. Th muttion i not inlun th ining o with NPHP protins. () Ovrxprssion o Vnus tgg rt in IMCD3 lls. Both n Q433R (grn) loliz to th ilium visuliz y o-stining o tylt tuulin (mgnt). Sl rs; 1 µm. Ntur Gntis: oi:1.138/ng.2681
![ANK Invs mrna lvls [%] 1 75 5 25 Invs-i - + Tt g positiv ili [%] 1 75 5 25 Invs-i - + Tt h Invs-i - + Tt γ- Tuulin Supplmntry Figur8.](/docs-images/91/107044019/images/8-4.jpg "Th nkyrin rpt omin o ANKS6 mits th intrtion with NEK8 () Shmti rprsnttion o ANKS6 truntions us or CoIP.")
8 Supplmntry Fig ANK SAM 1 - ANK SAM Flg-GFP Flg-CD2AP Flg- Flg-.ANK Flg-.SAM Flg-ANKS6 Flg-ANKS6.ANK Flg-ANKS6.SAM IP: nti-flg, WB: nti-v5 885 ANKS6 ANKS6.SAM ANKS6.ANK Flg-GFP Flg-INVS V5- Flg-INVS Flg-INVS IP: nti-flg, WB: nti-v5 Flg-Divrsin - V5- - Flg-Divrsin - Flg-INVS Flg- - Flg-CD2AP - Flg-INVS IP: nti-flg, WB: nti-flg Lysts: nti-v5 Flg- V5-GFP V5-GFP Flg-INVS V5- - Flg-ANKS6.SAM - Flg-ANKS6.ANK - Flg-GFP IP: nti-flg, WB: nti-flg Lysts: nti-v5 Flg-GFP Flg-NEK Flg-INVS Flg-GFP - V5- V5-INVS V5-.ANK -V5-INVS - Flg-INVS 1 - IP: nti-v5, WB: nti-flg - Flg- - V5-5 - IP: nti-flg, WB: nti-v V5-.ANK -Flg-NEK IP: nti-v5, WB: nti-v5 - V5-GFP 25 - IP: nti-flg, WB: nti-flg Flg-GFP -V5-INVS 1 - Lysts: nti-flg - Flg-INVS - Flg- 5 - Lysts: nti-v5 -V5-.ANK Invs mrna lvls [%] Invs-i - + Tt g positiv ili [%] Invs-i - + Tt h Invs-i - + Tt γ- Tuulin Supplmntry Figur8. Th nkyrin rpt omin o ANKS6 mits th intrtion with NEK8 () Shmti rprsnttion o ANKS6 truntions us or CoIP. () Flg tgg ull-lngth n trunt orms o rt n humn wr o-trnst with V5 tgg ull-lngth NEK8 in HEK293T lls. Atr immunopripittion with n nti-flg ntioy, NEK8 ws prsnt in th pripitts o th ull-lngth n th nkyrin rpt omin o rt n humn (ANKS6.ANK). It ws not tt in th pripitts o GFP, CD2AP (ngtiv ontrols) n th SAM omin o ANKS6 (ANKS6.SAM). () Flg tgg Divrsin n Flg tgg truntions o INVS wr o-xprss with V5 tgg ANKS6, ut ANKS6 o-pripitt only with th nkyrin rpt omin (AA 1-55) o INVS. () CoIP o wht grm lysts. n INVS wr prsnt in NEK8 pripitts. INVS lso intrt irtly with. () Flg tgg NEK8 ws o-xprss with V5 tgg INVS n th nkyrin omin truntion o. Atr immunopripittion with n nti-flg ntioy INVS ws only prsnt in th NEK8 pripitts whn ws o-xprss. () qpcr or Invs mrna o totl RNA xtrt rom Invs-i lls. Th knokown is inuil y ttrylin n rss Invs mrna lvls; rror rs: stnr vition. (g) Cili with positiv stining in Invs-i lls unr ttrylin inu onitions wr ru rom 1 to 2.7%, rror rs: stnr vition. (h) Wstrn lot o ovrxprss n Invs-i ll lysts wr stin or. protin lvl ws not ltr in th Invs knokown sitution (+ Tt). γ-tuulin srv s loing ontrol. Ntur Gntis: oi:1.138/ng.2681
![ili [µm] nk8 6 4 2 nks6 1.Tu Ho IMCD.-i +Tt.](/docs-images/91/107044019/images/9-4.jpg "Tu Ho 1.8 ili [%] IMCD.-i -Tt nk8 nks6 1.6.4.")
9 Supplmntry Fig.9.Tu Ho p5 WT 3months * p5 JCK 3months 8 lngth ili [µm] nk nks6 1.Tu Ho IMCD.-i +Tt.Tu Ho 1.8 ili [%] IMCD.-i -Tt nk8 nks Tt g nks6 nk8 h nks6 Cirulr stnr vition nks6 Supplmntry Figur 9. is not nssry or ili ormtion () Conol imgs o immunoluorsn stining o wiltyp n jk kiny stions riv rom p5 or 3 months ol nimls. (grn) loliz to th proximl prt o th tult tuulin stin ili (r) in wilttyp n jk (rrows). An ovrviw o img o rnl stions t 3 month inluing th DIC-hnnl (gry) is shown on th lt. Th strisk inits yst. Sl rs; 1µm. Pnls on th right show mgniition imgs without DIC. Th position o th mgnii r t 3 month is init y whit ox in th ovrviw img. Sl rs; 5µm. () Conol imgs o th ntrior sgmnt o th ilit pronphri tuuls o 24hp ol mryos, nk8 n nks6 morphnt mryos. Sl rs; 1 µm. () Quntiition o th iliry lngth in pronphri tuuls. Ciliry lngth ws not t y injtion o 2ng nk8 or 2ng nks6 1. () knokown os not t ili ormtion in IMCD3 lls. Cili r visuliz y tylt tuulin (grn) n nuli with Hohst (lu). Sl rs; 1 µm. () Quntiition o ilit lls. In th knokown sitution (+Tt; light gry rs).85% n in th non-inu lls (-Tt).74% o lls r ilit (rk gry rs); rror rs: SEM. () Snning ltron mirosopy (SEM) piturs o ilit pirml lls rom Xnopus mryos tr injtion into oth vntrl lstomrs. Knokown o nks6 n nk8 i not t ili ormtion in omprison to ontrols. Sl rs; 1 µm. (g) Dpltion o nks6 in th Xnopus pirmis rsult in mil prturtion o sl oy polriztion, s rvl y th loliztion o CLAMP-GFP n Cntrin-RFP, mrkrs or rootlts n sl ois rsptivly. Whit oxs init th mgnii r on th right pnls. (h) Th polriztion ws quntii y ngulr msurmnts o CLAMP/Cntrin pirs. iiny rsult in n inrs o irulr stnr vition s ompr to ontrols; ( p<=.1); Mnn Whitny rnk sum tst; rror rs: SEM. Sl rs; 5 µm. Ntur Gntis: oi:1.138/ng.2681
![Motiv irn kiny lngth uninj -inj si [mm].6.4.2 ** * 42 36 2 hi1n 5 hi1n +.1ng +.](/docs-images/91/107044019/images/10-3.jpg "3ng HIF1AN g 25-5 - 25-15 - 1 - m ormtion [%] pdna3 IP: nti-flg, WB: nti-v5 - V5-NPHP3 - V5-INVS - V5-CD2AP - V5-NPHP1 - - V5-Lui - V5-HIF1AN - V5-GFP Lysts: nti-v5 - Flg- IP:")
10 Supplmntry Fig.1 V5-HIF1AN Flg Flg-GFP Flg-INVS Flg-NPHP3 Flg-NEK8 Flg- IP: nti-flg, WB: nti-v5 Flg-Divrsin - V5-HIF1AN - Flg-NPHP3 - Flg-INVS - Flg- - Flg-Divrsin - Flg-NEK V5- Lui/ -CD2AP/ -GFP V5-NPHP1,3, INVS, NEK8 + V5-Lui V5-NPHP1,3, INVS, NEK8 + V5-HIF1AN - V5-NPHP3 - V5-INVS - V5-NPHP V5-HIF1AN IP: nti-flg, WB: nti-flg Lysts: nti-v5 - Flg-GFP - V5-HIF1AN CADALLKAGADVNKTDHSQR 82 humn INVS CADALLKAGADVNKTDHSRR 82 mous Invs CAEALLKHGAQVNRSDRSGR 59 X.trop Invs VAHLLLDHGADVNAQNRLGA 145 humn ANKS6 VAHLLLDHGADVNAQNRLGA 136 mous VAHLLLDHGADVNAQNRLGA 136 rt VAMILLENGADVNAQNRLGA 147 X.trop VVRLLMEWGADPNHAARTVG 225 VVRLLMEWGADPNHTARTVG 216 humn ANKS6 mous VVRLLMEWGADPNHTARTVG 216 rt VVRLLLDWGADVNYRVKTTG 246 X.trop VVKLLLEAGADVNAKDKNGR Hyroxl. Motiv irn kiny lngth uninj -inj si [mm] ** * hi1n 5 hi1n +.1ng +.3ng HIF1AN g m ormtion [%] pdna3 IP: nti-flg, WB: nti-v5 - V5-NPHP3 - V5-INVS - V5-CD2AP - V5-NPHP1 - - V5-Lui - V5-HIF1AN - V5-GFP Lysts: nti-v5 - Flg- IP: nti-flg, WB: nti-flg ng 16ng hi1n N129AN29A pdna3 + CoCl2 N129AN29A Hi1α γ-tuulin Supplmntry Figur 1. Th untion o HIF1AN () CoIP o V5 tgg HIF1AN. HIF1AN ws oun in th pripitts o INVS, n ut not Divrsin. () CoIP xprimnts o Flg tgg rt n V5 tgg NPHP1, INVS, NPHP3 n HIF1AN. Atr immunopripittion with n nti-flg ntioy NPHP3 n INVS protin lvls wr inrs in pripitts whn HIF1AN ws o-xprss. () Alignmnt o HIF1AN onsnsus squn rom INVS n ANKS6 orthologus o init spis. Gry unrly rsius rprsnt onsrv mino is o th rognition moti with ssntil rsius in r. Th hyroxylt sprgin (N) is shown in ol. Th numrs on th right init th position o th C-trminl mino i pit. () Biltrlly hi1n injt Xnopus mryos vlop m in ontrst to th ontrol mryos. Sl rs; 5µM. () Quntiition o m ormtion in prntg. () Quntiition o hi1n rsu xprimnts. Th irn o th kiny lngth [mm] rom th uninjt to th injt si oul rsu y o-injtion o HIF1AN RNA (* p=.5; ** p=.2; p=<.1). t-tst; rror rs: SEM. (g) Atr trnsint trnstion HEK293T lls wr trt with CoCl2 or 8 hours. Th lysts wr prpr n nlyz or Hi1α lvls. Ntur Gntis: oi:1.138/ng.2681
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