Journal of Cell Science o:10.1242/jcs.131250 doi: 3770–3781 126, Science Cell of Journal 2013 May 15 Accepted n aeo eetr.K8lne oybqiyaintg proteins tags polyubiquitylation K48-linked receptors. of fate and fusion after The degradation lysosomal signaling. lysosomes. by with cytokine cleared gets attenuating content MVB thus cytoplasmic intraluminal from molecules, away into receptor signaling the membrane sequesters outer MVBs of the vesicles via from degradation of the receptors Incorporation lysosomal (MVB). to activated bodies for back multivesicular out or recycled al., endosomes sorted be et late or (Clague either station membrane can sorting receptors plasma central here, a as From by serve 2012). platform and 2012) ligand (Pa signaling al., pathways signaling a et of specific to as irrespective receptors operate activated coupling endosomes, endosomes early These binding. into internalized cell are Following the routing. at intracellular novo subsequent exposed the surface. their on receptors cell and depends surface the signaling-competent additionally at cytokine of receptor a its number to to response cytokine cellular initiated the The transcription of gene binding which and 2008), after transduction al., signal et of the (Croker suppressors suppress proteins and 2005) (SOCS) activated Liu, signaling of and like cytokine inhibitors (Shuai Many mechanisms proteins protein (PIAS) 2008), inhibition signaling. STAT Qu, feedback and pathological (Xu on phosphatases focused potentially controlled have tightly aberrant, is studies signal avoid cytokine a to to sensitivity cellular The Introduction for sorted are receptors whether and USP8 determines of thus levels. regulator receptor USP8 key cytokine words: and a basal Key as regulating RNF41 way acting of this by recycling, cross-regulation trafficking our or Collectively, inhibitory receptor reciprocal degradation USP8. leukemia In cytokine lysosomal of Balanced and levels. suppression JAK2-associated leptin USP8 through stability. model controls reduces complex the RNF41 ESCRT-0 ESCRT-0 and on the that USP8, expression destabilizes demonstrate that ubiquitylates ectopic indirectly reveals RNF41 and findings RNF41 and of Moreover, redistributes RNF41 effects receptors. of RNF41 the substrate (LIF) effects. a matches factor as RNF41 functionally USP8 JAK2-associated knockdown aforementioned identifies and USP8 of the study RNF41 addition, present shedding explains stabilizes The to ectodomain USP8 (DUB) proteins. enzyme enhances of transmembrane ubiquitin response various deubiquitylating and E3 loss of cellular RNF41-interacting the degradation trafficking an that in lysosomal the (USP8), involved reported basal 8 is consequently previously inhibits protease We and Ubiquitin-specific (RNF41) of shedding. receptors. 41 receptors, balance ectodomain cytokine protein dynamic cytokine and finger a degradation of RING is recycling, receptors ligase levels internalization, surface-exposed membrane, of surface plasma number cell The the understood. steady-state poorly remain the cytokines, controlling mechanisms The Summary work this to equally ` contributed authors *These 2 1 processing Ceuninck De and Leentje sorting receptor cytokine USP8 and controls RNF41 between cross-regulation Reciprocal 3770 uhrfrcrepnec ( correspondence for Author eateto iceity aut fMdcn n elhSine,GetUiest,Abr arseka ,B90 hn,Belgium Ghent, B-9000 3, Baertsoenkaai Albert University, Belgi Ghent Ghent, B-9000 Sciences, 3, Health Baertsoenkaai and Albert Medicine (VIB), of Biotechnology for Faculty Institute Biochemistry, Interuniversity of Flanders Department Research, Protein Medical of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. bqii evsa rfikn inladmdae h sorting the mediates and signal trafficking a as serves Ubiquitin ytei n eieyt h lsammrn receptors membrane plasma the to delivery and synthesis ahpi- laae rp,RF1 ye1ctkn eetr S8 cooanshedding Ectodomain USP8, receptor, cytokine 1 Type RNF41, Nrdp1, cleavage, Cathepsin-L [email protected] 1,2, ,JrsWauman Joris *, ) 1,2, ,Dlhn Masschaele Delphine *, ´ lfy de ysriga cfod -b,ND4 SOCS3, NEDD4, C-cbl, scaffold. substrate a specific as E3 a serving to (E2) by Several enzyme lysosomal the ubiquitin-conjugating transfer cargo. a and ubiquitin from coordinate endocytosis of They of the receptors. cytokine in regulation of targeting traffic involved are allows intracellular ligases ubiquitin proteins and adaptor recruitment endocytic the and and these for other (Raiborg of ubiquitylation degradation essential and Phosphorylation lysosomal are 2009). towards Stenmark, proteins cargo (ESCRT) of selection transport for complex ubiquitylatedrequired endosomal their The pathway. mediate recognize endocytic and the along (UBD) proteins progression domains ubiquitin-binding Adaptor via 2011). proteins Piper and 2009; Lehner, endocytosis (Komander, receptors the and various in of delivery implicated lysosomal are monoubiquitylation and polyubiquitylation K63-linked while degradation, proteasomal for as nw sNd1o LF,afcstebslruigof routing basal the affects FLRF), or Nrdp1 as receptor known cytokine (also govern stimulation. cytokine that of absence mechanisms the in the trafficking about Wo known 2008; 2012; al., et Kumar al., Tanaka 2004; al., 2012; et et Kumar Malarde 2011; Almeida 2007; al., al., et et Silva Jiang 2005; (da al., et Gesbert complexes. gp130, and receptor interferon, (IL-10) interleukin-10 internalization I and growth (G-CSF) type factor ligand-dependent erythropoietin, colony-stimulating common, granulocyte activated gamma in prolactin, the hormone, role of degradation a lysosomal play CHIP ercnl eotdta h 3uiutnlgs RNF41 ligase ubiquitin E3 the that reported recently We 1,2 rn Peelman Frank , ´ ta. 09 ee ta. 07 lta tal., et Slotman 2007; al., et Meyer 2009; al., et llre l,20) oee,ltl is little However, 2009). al., et ¨lfler enovo de 1,2 n a Tavernier Jan and ytei,tasotto transport synthesis, eerhArticle Research b TC and -TRCP 1,2, um ` Journal of Cell Science rnmmrn DM( iitgi n metalloproteinase) by and shedding disintegrin ectodomain (a their ADAM formation, enhances CTS transmembrane receptors, these simultaneously blocks of RNF41 degradation and lysosomal lysosomes. reduced the reflects (LIFR which in receptor degraded LIF is (LR), receptor (IL-6R receptor leptin the L cleaves cathepsin that observed We receptors. cytokine JAK2-dependent rti ees(a ta. 07.Cnesl,etpcexpression ectopic Conversely, RNF41 2007). stabilizes al., et and (Cao RNF41 ubiquitylation levels ligase protein ubiquitin RNF41 E3 suppresses the of USP8 substrate a is USP8 Results shedding. and of degradation role functional receptor the cytokine for in accounts RNF41 thus USP8 ectopic of RNF41 Loss of expression. effect the of In resembling degradation phenotypically shedding, USP8. LIFR lysosomal and relocalizes reduces LR the knockdown USP8. and and USP8 destabilizes RNF41 addition, We between ubiquitylates, 2007). regulation al., reciprocal RNF41 et a (Cao reveal ubiquitylation here RNF41 RNF41 al., stabilizes reverts USP8 et and Moreover, the (Berlin levels 2007). al., recycling et receptor directs Niendorf favors 2010b; growth (EGFR) epidermal USP8 receptor the factor of complex, degradation, deubiquitylation lysosomal USP8-mediated towards ESCRT-0 2006). while (CXCR4) ESCRT-0 al., 4 en et the receptor Row trafficking the chemokine 2007; receptor stabilizing al., et of constitute (Niendorf By steps growth-factor-regulated lysosomes early the together to the route hepatocyte govern which and complex and (Hrs), and substrate STAM (STAM) molecule USP8 various adaptor proteins. stabilizes transducing sorting of signal and cargo with modulating degradation both interacts by of the endosome dynamics ubiquitin sorting regulates the the USP8 at 2011). Ubpy) proteins al., as transmembrane et (Wright known determines sorting (also and lysosomal ubiquitylation of stability efficiency between protein the balance regulates deubiquitylation The direct and ubiquitylation. the be protein might of trafficking. receptor proteins cytokine in these assist of and RNF41 one of complex, substrate As that ESCRT the to complexes. speculated coupled (Markson both receptor USP8 2004), we al., and the et cytokine TSG101 Wu 2009; with I al., influence interact et to type not shown these Yu was RNF41 does 2009; LIFR all al., to however LR, et common RNF41 Wang of 2004; 2008). ubiquitylation al., Zhou, et Qiu Qiu and 2008; 2002; al., Goldberg, et (Jing and receptors erythropoietin (RAR and alpha interleukin-3 receptor acid retinoic unspecified ErbB3, and of polyubiquitylation TBK1 of K63-polyubiquitylation induces BRUCE, it and while Parkin MyD88, ubiquitin RNF41 of E3 K48-polyubiquitylation substrates. (RING) mediates various gene ubiquitylates new differentially interesting and really ligases of crossroads family the the to cellular belongs RNF41 at or occurs. way shedding lysosomes ectodomain where acts towards this compartments routing thus receptor, receptor cytokine membrane-bound RNF41 between respective trans-signaling. the enabling cells of stimulation lack cytokine allow that receptors by soluble IL-6, signaling of in Moreover, case benefit 2008). 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(CTS) oso erimn fteE ojgtn nye(i and RNF41 endogenous (Qiu and enzyme mutant to this conjugating due between expression E2 to competition USP8 direct stabilizes due the strongly activity also of 2002), ligase Goldberg, recruitment ubiquitin additional as E3 of to impaired referred loss sequestering (further with mutant SQ), H36Q C34S RNF41 by RNF41 Expression Etagged suppression. counters an USP8 of activity for mutant it necessary this factors although likely, endogenous RNF41 Most levels, USP8. AE USP8 on endogenous with RNF41 depends endogenous 1C), interacts The (Fig. RNF41 enhances hardly USP8. and by even levels RNF41 USP8 mutant between protein antibody of interaction an USP8 suppression direct by that endogenous recognized no indicating it expression lowers readily mutant as AE is longer RNF41 functional RNF41. and and endogenous proteins shown) detecting in folded RNF41-interacting not USP8 other properly with (data to interaction is the binding mutant preserves The reduced AE 1B). (Fig. strongly RNF41 assays GST-pulldown with and to AlphaScreen referred AE) MAPPIT, (further 2006), mutant C-terminal RNF41 R269E al., the Q266A et as RNF41 with (Avvakumov an RNF41 created complex the in of we of in domain USP8 structure USP8 binding crystal endogenous substrate of the of on domain levels Based Rhodanese 1A). protein (Fig. the cells HEK293T lowers RNF41 of el.Hwvr oepeso fRF1rdsrbtsUP to USP8 1E). (Fig. redistributes vesicles RNF41 USP8. intracellular still of large of co-expression mutant HeLa RNF41 However, localization throughout AE cells. distributed homogeneously Finally, intracellular is RNF41 USP8 the ubiquitylation. Exogenous the changes USP8 why dramatically provokes explain USP8 enhances with moderately might expression interaction Residual 1B) SQ (Fig. 1D). RNF41 (Fig. to not ubiquitylation lysosomal USP8 USP8 chloroquine but or was and exogenous proteasomal RNF41 This MG132 preceding degradation. RNF41. both of linkage of of ubiquitin presence analysis stabilize substrate the a ubiquitylation in performed is by levels ubiquitin USP8 USP8 E3 confirmed reduce that the to suggests indispensable that is reveals and RNF41 This of 1C). activity (Fig. ligase binding USP8 for epeiul eosrtdta N4 xrsinblocks expression RNF41 LIFR LR, that L-mediated demonstrated cathepsin previously LIFR and We LR cathepsin of for degradation necessary L-dependent is expression protein USP8 ´ 06.RbGPsscoordinate GTPases Rab 2006). , a n IL-6R and a a -emnlstub C-terminal Journal of Cell Science otat-tgadascnaydne niga lx lo 8 nioysann.RF1c-xrsinwsvrfe sn rmr os anti-RNF4 10 mouse bars: primary a Scale using shown). verified (not was staining co-expression RNF41 antibody staining. 647 antibody Fluor 488 Alexa Fluor anti-mouse Alexa anti-goat donkey donkey secondary secondary a and anti-Etag goat pMET7-solIL-5R or vector RNF41 nii n rtaoa rlssmldgaain LGUP a muorcpttd(P n t bqiyainsau a sesdb etr blo western by assessed was status ubiquitylation its and (IP) immunoprecipitated was FLAG–USP8 ( degradation. 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Etag–USP8 SVT different encoding or the plasmids mutant of with levels co-transfected AE expression transiently the were for cells correct HEK293T to AlphaScreen, receptor, bait the with associated obndwt h X22rA1lcfrs eotr etdy el eesiuae ihEofr2 or rlf nrae.Lcfrs ciiyis activity Luciferase untreated. left or hours were constr 24 cells prey for pMG1-USP8 HEK293T Epo FLAG-tagged analysis, or with empty MAPPIT stimulated an were For stimulated) with cells (stimulated/non assays. together day, induction bait (right) Next mutant fold GST-pulldown AE reporter. RNF41 pXP2d2-rPAP1-luciferase and or the (middle) RNF41 with wild-type AlphaScreen combined a (left), encoding MAPPIT plasmids with in transfected binding transiently USP8 impedes AE) (RNF41 i.1 S8i usrt fteE bqii iaeRNF41. ligase ubiquitin pMET7-solIL-5R E3 with the transfected cells of in substrate that a to compared is USP8 1. Fig. 3772 C ora fCl cec 2 (16) 126 Science Cell of Journal coi xrsino h N4 Emtn rteEtge N4 3SH6 RF1S)mtn aldt eueedgnu S8lvl in levels USP8 endogenous reduce to failed mutant SQ) (RNF41 H36Q C34S RNF41 E-tagged the or mutant AE RNF41 the of expression Ectopic ) D E N4 nacsUP bqiyain E23 el otasetdwt MT etr noigFA–S8 Auiutnadeither and HA–ubiquitin FLAG–USP8, encoding vectors pMET7 with co-transfected cells HEK293T ubiquitylation. 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(mock). a a H and –HA C C- (C) Renilla Journal of Cell Science sn etr blotting. western incubated 25 using and without serum-deprived or were with or Cells overnight USP8 C786S. wild-type USP8 encoding inactive plasmid catalytic FLAG-tagged of levels increasing solIL-5R of irrelevant effect LR–HA, inhibitory encoding the reverses formation. USP8 CTS on of RNF41 expression Ectopic 3. Fig. before reported as 3), lane enhances versus USP8 5 wild-type lane 3, hand, (Fig. other stability the RNF41 on while suppression 1A,D), and (Fig. ubiquitylation other’s USP8 each induces influence self- RNF41 RNF41 lane expression. and 3, USP8 by Importantly, (Fig. of 5). 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CAAACCACTTTGTACAAG-3 3 GATGACGACGATAAGGAATTCTCGACCGTGGTAC-3 GGTCGAGAATTCCTTATCGTCGTCATC-3 MT-ARb1S5 ih5 with S25N pMET7-HA-Rab11 Lvn ta. 06.HmnRF1wscoe rmtepMET7-Etag-hRNF41 the from cloned was RNF41 a Human into 2006). al., ligated et (Lavens was fragment PCR TTTCCAACACCAGAATC-3 GCTGTCTGATTTACTG-3 EadRF1A atwt N4 26 29 uainusing mutation R269E Q266A RNF41 5 with bait 3 5 AE and RNF41 5 and AE GGCGTTGCAGAAAGCTTGTTCACTATGAGGTGCCTGTAC-3 LGUP 76 ih5 with C786S FLAG-USP8 rvddb oh,it nepypE7vco.Alcntut eevrfe by verified were constructs All vector. pMET7 analysis. empty sequence an DNA into Roche, by provided of ligation via 5 9 9 9 9 9 9 9 MT-N4 QEa ihRF1C4 3Qmtto using mutation H36Q C34S RNF41 with SQ-Etag -GTACAGGCACCTCATAGTGAACAAGCTTTCTGCAACGCC-3 pMET7-RNF41 ; -GCCACACTAGAGACTCGAGCGATGAACGAACGCTACTATGAGAAC-3 -ATTGAGTTCATATAACTAGTATTTC-3 -CGCTCTAGATCAGCAACTGCAGCTTTCTGCCGAGGCCTTGG-3 -CCCCAATTGACAAGTTTGTACAAAAAAGC-3 ˚ n rw nDlec’ oiidEgesmdu DE)(Invitrogen) (DMEM) medium Eagle’s modified Dulbecco’s in grown and C I ie;Rb1 sn 5 using Rab11A site); I pndpG-V etr(ykra ta. 01 with 2001) al., et (Eyckerman vector pMG1-SVT opened I n 5 and -Not 9 9 9 -GAGTGAATTCTTCCCGACTCCAGAATCTCC-3 -CGCGCGGCCGCTTATATCTCTTCCACGCCATGCG-3 -GTTCTCATAGTAGCGTTCGTTCATCGCTCGAGTCTCTAGTGTGGC- 9 9 -ACAAAAAAGGCGGCCGCATGCCTGC-3 ieto ftepE-KP2vco Ecemne l,20) The 2001). al., et (Eyckerman vector pSEL-FKBP12 the of digestion I ognrt h MT-LGRF1vco.pMET7-FLAG-Rab4A, vector. pMET7-FLAG-RNF41 the generate to Mfe 9 -AATAAACTGATGAAGTAAGCAATTTTTCCCAGTTCCTGCA-3 9 9 I -TGCAGGAACTGGGAAAAATTGCTTACTTCATCAGTTTATT- -Xba Eco 9 9 (with 9 RI n 5 and ieto h is C rgetwslgtdit an into ligated was fragment PCR first the of digest I -GCGGAGCTCGATGGGGTATGATGTAACCCGTTTCC-3 (with -Xho Not 9 N4 E eie rmteRF1A at was bait, AE RNF41 the from derived AE, RNF41 . m 9 u oul IL-5R soluble cut I N rnfce.O h etdy el eewashed were cells day, next the On transfected. DNA g 9 9 notepDA. etrusing vector pCDNA3.1 the into , ie n 5 and site) I 9 -GGCCGCCTTATCGTCGTCATCCTTGTAATCCA- -TCTGGAGTCGGGAAGAATTCACTCATGAAC-3 9 Eco n 5 and (with 9 MT-N4 E-tg,pMET7-FLAG-RNF41 AE(-Etag), pMET7-RNF41 ; 9 -TAACTTAGGAAATACTAGTTATATG-3 Ist)ad5 and site) RI 9 9 9 -GATTATGCTGCGGCCGCCATGGGCACCC- 9 -GCGGAATTCATGGCTAGTCGAGGCGCAA 9 Not 9 n h M1pe etnto etra a as vector destination prey pMG1 the and -AATTCCATGGATTACAAGGATGACGAC- -CGAGTAAATCGAGACAGCAGGTTATTC- Xba 9 Sac -GATTCTGGTGTTGGAAAGAATAACCT- (with ieisre yst-ietdmutagenesis site-directed by inserted site I 9 ie.TePRpout eeligated were products PCR The site). I Not epcieyad5 and respectively , II -Xba Sal I 9 -Xba -CAGTCTCTAGACAC-3 9 9 Xba -GTTGTCTCTAGATTAGATGTT- I a pMET7-solIL-5R . 9 pndpE7Ea-i vector pMET7-Etag-Cis opened I -Not . rmthe from Sac ,dwsra fguahoeS- glutathione of downstream I, ie;RbAuig5 using Rab7A site); I Eco ,gnrtn h GST-RNF41 the generating I, II 9 9 -CGCTCTAGATTAGTTA- Xba 9 -Xba 9 2 RI n 5 and uiiidamshr at atmosphere humidified -Not (with r5 or iewsgnrtdin generated was site I l 9 ieto h second the of digest I 9 gt11-hIL5R pMET7-FLAG-Rab4 ; n 5 and n elcdb a by replaced and I 9 Not 9 -GCAGGCATGC- -GGGTCTAGAT- Eco 9 Bgl a -CCCCCGCGG- 9 I 9 -Xma pMET7- ; a generated was 9 Ist cloned site RI ihpMET7- with Ist)and site) II -GTACCAC- 9 pMET7- ; 9 9 9 a .PGEX- I. 2vector 12 n 5 and n 5 and 9 n 5 and Eco 9 6 (with and 10 RI 9 9 9 9 9 9 9 9 5 - 9 - 9 - - - - - ; Journal of Cell Science muto rnfce N n odo h rncitoa n translational and transcriptional the of load and DNA machinery. transfected of amount ih3 with fpE7Ea-S8pamdwt rwtot50n fpE7hN4 vector. pMET7-hRNF41 pMET7-solIL-5R ng of the 500 ng experiments, with 500 all without guidelines In or transfected manufacturer’s with were the plasmid cells pMET7-Etag-USP8 to of day, according next (Polyplus) JetPrime The with (Sigma-Aldrich). poly-D-lysine with coated E23 el eesee n1-el n rnfce ntenx a with day next the on transfected and 12-wells in seeded were cells HEK293T ubiquitylation 1 For with phosphate. day calcium next using the 2.4 proteins on assays, transfected FLAG-tagged and and E- 6-well and 3 both a bait analysis, of in AlphaScreen ng seeded For 250 were phosphate. reporter, cells calcium pXP2d2-rPAP1-luciferase HEK293T using of constructs ng prey 50 ng with 250 day next the on n a ottaseto,clswr ahdadtetdoengtwt 25 with overnight treated and washed were cells post-transfection, day One assay Ubiquitylation 7.5, pH Tris–HCl mM (50 MgCl mM buffer 1.5 lysis NP40, TAP 0.2% glycerol, in and 5% later day NaCl, manufacturer’s next hours mM the the the 24 125 on with washed another to vectors, overnight expression lysed co-transfected according FLAG-tagged were and conducted E- cells appropriate Typically, were (PerkinElmer). experiments protocol possibly screen for corrects Alpha which prey, AlphaScreen binding used. baits JAK2 different a the was of by samples levels generated expression triplicate varying relative signal units) and from the light (PerkinElmer) relative activity to luminometer (stimulated/non-stimulated induction TopCount hours fold Luciferase 24 a as expressed for in ng/ml). stimulated chemiluminescence (5 or by Epo measured untreated left human were cells with transfection, overnight After analysis MAPPIT 5% glycerol, 10% SDS, 3% 2 6.8, in pH lysed PBS, HCl with washed were Cells analysis blot Western 25 with overnight treated or (DMSO) untreated post- left day were One method. cells ng phosphate 500 transfection, calcium with the together using plasmid plasmid mock/RNF41-expressing mock/Rab-expressing ng 500 or vectors encoding 1 yae eecerdb etiuainadicbtdfr1hu t4 at hour 1 for incubated and centrifugation by cleared were Lysates Na PriEmr.Pr fteclyaewssmlaeul nlzdfrprotein for analyzed simultaneously was cellysate blotting. platereader western the EnVision using the expression of using triplicate Part in (PerkinElmer). measured were Samples respectively. el eewse ihPSadlsdi 250 in lysed and PBS with washed were Cells 5 and chloroquine in detection. 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Alexa scanning 60 donkey anti-mouse a laser with using donkey temperature IX-81 four acquired room and were at After Images 488 hour antibodies. antibodies. 1 Fluor further Cruz) for Alexa were incubated Santa were Samples anti-goat (1:500, cells hour. (1:4000, A-6 PBS, A190-132A 1 in anti-RNF41 anti-Etag washes for goat mouse with serum in temperature and blocked donkey room Bethyl) at and whole hour minutes cells and 1 5 PBS, for for BSA incubated with PBS 1% in washes with X-100 at three Triton minutes PBS After 0.4% 15 with for paraformaldehyde. permeabilized fixed 4% and were PBS in with temperature rinsed were room cells post-transfection, hours 24 microscopy Confocal D-005203-03 siGENOME siRNA2 USP8 D- 1.25 siGENOME or (Dharmacon). siRNA1 or USP8 (Dharmacon) (Dharmacon) using accomplished 005203-02 was were knockdown Transient Cells silencing (BioVendor). USP8 western kit using analysis ELISA cleavage (CTS) sLIF-R/gp190 stub ELISA C-terminal blotting. human based for leptin-SEAP lysed the developed simultaneously in-house LIFR or or an LR using soluble method and determined collected were was supernatant levels cellular day, Next (Invitrogen). ed G elhae o ora 4 4B at Sepharose hour Glutathione in 1 proteins on for GST immobilized soluble Healthcare) the were (GE obtain proteins to beads GST minutes 10 supernatant. for the r.p.m. 12,000 at centrifuged n vlaino iecn fiinyuigwsenblotting. analysis western cleavage using (CTS) efficiency stub silencing C-terminal of ELISA for by evaluation lysed receptors and simultaneously soluble detect were to using 25 cells collected incubated without and LIFR was were or supernatant or cell with cells LR morning, medium transfection Next the OPTIMEM siRNA serum-free encoding following in plasmids hours overnight or 72 plasmid phosphate. control calcium a with transfected 50 oricbto ihSpaoe4 ed,rttn t4 at rotating beads, 4B Sepharose with incubation 20,000 hour at 1 centrifugation minutes 30 r ahdtretmswt ENbfe.FA-agdpoen on othe Coupled to blotting. bound western Quick 2 and proteins in SDS-PAGE FLAG-tagged boiling T7 by minutes buffer. analyzed 10 TNT NETN by beads eluted with the were the times GST-proteins and three overnight using washed added are was produced (Promega), System USP8, Transcription/Translation FLAG-tagged buffer. 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Renilla ih1m fws ufr(0m rsHl H80 NaCl, M 1 8.0, pH Tris-HCl, mM (10 buffer wash of ml 1 with 6 10 5 E23 el na1-elwr eeetasetdwith transfected revere were 12-well a in cells HEK293T uieaesRAP027-12 sangtv control negative a as P-002070-01-20 siRNA luciferase 2 MET;05 P0 %DTand DTT 1% NP40; 0.5% EDTA; mM 1 ; g ˚ ne oain yae eecerdby cleared were Lysates rotation. under C n h uentnswr rcerdby precleared were supernatants the and ˚ 6 n ahdtretmswt NETN with times three washed and C D e odn ufr ape were Samples buffer. loading gel SDS 6 .5N betv na Olympus an on objective NA 1.35 6 D e odn ufrand buffer loading gel SDS ˚ .Atrovernight, After C. .coli E. m chloroquine. M yaewas lysate ˚ ,beads C, Journal of Cell Science aln . ui,C .adHris .W. L. Harries, and E. C. Rudin, K., Hanlon, eWt . ihesen . el,R . ez,H . lmemn .advnder van and J. Klumperman, J., H. Geuze, B., R. Kelly, H. Y., S. Lichtenstein, G. H., Wit, A. de Rossum, van and J. G. Strous, C., A. Almeida, Silva da E. S. 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