IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. Cell (0092-8674) 2010 imex curation Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions tetth Tetrahymena thermophila pull down pull down sequence tag sequence tag identification sequence tag Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at Accepted 2010-JUN-24 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. Cell (0092-8674) 2010 imex curation Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at Accepted 2010-JUN-24 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. Cell (0092-8674) 2010 imex curation Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at Accepted 2010-JUN-24 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. Cell (0092-8674) 2010 imex curation Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions theth Thermus thermophilus anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at Accepted 2010-JUN-24 by ORCHARD. 25-Jun-2010: Contacted by JYOTI. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. Cell (0092-8674) 2010 imex curation Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at 25-Jun-2010: Contacted by JYOTI. orchard Only protein-protein interactions theth Thermus thermophilus anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Noto T., Kurth HM., Kataoka K., Aronica L., DeSouza LV., Siu KW., Pearlman RE., Gorovsky MA., Mochizuki K. Fig 1 (et al) experiments use the term "coimmunoprecipitated" several times rather "comprehensively" - nothing I can annotate. kazufumi.mochizuki@imba.oeaw.ac.at Accepted 2010-JUN-24 by ORCHARD. Cell (0092-8674) 2010 orchard Only protein-protein interactions imex curation b8xqc5_tetth WAG1 protein protein tetth Tetrahymena thermophila MEVSLVNQEMEQKDDISIESGQLSCEQLDAAKDINHMIQNKQPALDDKEAEDEEPKSFVFEEINTDRVQQILQEKQNEKPKKQSSGEKDSMHQNGNEDQPLRQLQSDMRTDDSKPLRESANTQVDFAKMSNCSTTWESNTKDETSITQNQSISNTSGSGWGGQSQNELVPKSDSSSNIWGGSQNSMGAQTESKSSSSGWGSSNKDKVNNESVQSSSTGGWGAQSNDNTSSQNNEGNGGGWGSSTNNNPSSSTAVWGSSTSGNEDNSNKNNSEGGWGNQSTDSGGWGSSNGNGSNNNNNNNNSGGGRFNRNNNNNNGNNGGGRRFDRNNNNGESGNDNNGGQRRFNNNNNNRNFNKEGGENKNGNNGGGWNKNNNNRNGGERNNNGGGFQNQNRNNSFKKTNSNEGQSNNSGGWNSQSNDNSQSTGGSGWGASSNDANQNNTTTSSSGWGSSTNENKNSSEASSGWGSSNTNTTANTNQTSESSGGWGSQQTENKQESQSSSGGWGNTSDQTQGGGDSWGSTSTNNNNNSNTNNNNRFRNNGNNNQNNNNNRQGGNRAWGQNKDQQGGKQGNGSWGNSKQNNEQQANKSDSQDGWGNNSSNNENSTTSGGWGNQESSKIENSTQSGTGGWGSSFSNNNNSSSENNGGGNNGKKWGASDNQNQGENKRQGSWGNGNNRNNSNQNQGGERQNFRGNNNRNNNNNNSSFVNNNKQKSNSGEGGWNNSSDNAPSVQSGGGGWGSTTEDNTTVQGGCGGGWGSSQVKEAASTQATTTSSWGGNNQDSLNNDSKLLTKRPSKDPRLQEKKQTQVLSINDMSETEVQERIFGTKRVKKNEEGEDSILQAKSKIPQFIDLEKEEEGCFARFSPLIVDRDTDKEYNPTGIPLDEQIYEGSINQLISNKDTIDQEDGDDDDLEMAIIINPQKLLESQLKKYQTYSGFFQVYKKYLVPEKQSILCEPCENTAYNLFHDRLVYVIPDEPKRYNILCQFNTFMPYEQYRHLPLISTRNRTSQQELGFLDYETAIESIKNIQIQETKERRTIFPCWVESSLPNQHVELNKLAKYLLDKNMVAGYNYTNHATINLMHVKQIFPDLEYVELPEQSENQKTSLICLNTLEASIYDSFCQKIPAYE 399536F77272AAAA q22a77_tetts TTHERM_01276320 protein protein tetts Tetrahymena thermophila (strain SB210) MQSFVNLIQLEENYNLSLLSYSNSIRQYLLDNKVHEDKYIQLMKDIKDLFSSPTFLMPNFSDKRKQFFSQLCLIAFDFANNQVTEQQSLSEFRKKFKFPFVIIAIIYTKLLEIKEALPEDFLTLVDCHFKNKGPQSIFASLTLISLFLNEVDQYEIDCDHIESSNTLSSCSEAAESNIPKKVNITVKTVQNFFDKQGLQMIAFPLNVIKNIHCVAHDQQSIIQLLLKIVVIIIKNMSNKIKQEFFYFHQKDLNVILSCFIGFLQQNNQAREDRDVALANIFEFCELLAKSTSPAQLYVAFKDFLVYISQNFSFANDSIEYFDFNSKCVDFLVSITPCFIQQSENLSAYPLIIQLLKQSAKINLVNLEDQIGCLHIWSHYSDQFIQAYDNNNNSIKAETINNLAILNSTFIESLQFDIELLFNLQIKDHDSEDVNQFVDQLSRLVIHSNLIDNQLFVEVIKSLSLLYQKNMLLYYTFQKCLMKQYLNSKDTLSQIYQVFGKSNDFLQITELLFESFSQAQKQFANLDLNTIAAIEKFTQFMSQIGAKLFNTQISHLEQSQILGIFQKYILNPIKMFNLNIFSQLPENFIQLNEVQFSQVCTMMESVHYFIFSDNKSNSDQVFETEVDVSMIKKILLFNQSVNQDMAYWINPQLKNERKKIKQFYQSTIYWVIFASINLENAANIFESYFFQKEFNIWVLNSTIKKLKGRILPNTFLLYLCYYLKSLTDSDSFSKIVQKSEYEEKFRKLMVNISKSVPSEIYVDYSKIQYYLQLYINEECQMFRFQILSDQTVSLLTLENRFSHVYKVFIILEDLFTIVQRFPKFYYENTFMGLKRNFFLNRVFWIIVKCFTIVPKQELLENSQIYKTISLYLNLAIRFKMDFLEKEETQSENSHYFNFILEILDHMYFTNKKIVLDFCQKILNYFDQDCYNQNIIQLAQSSQTFQNITLQLFTNIWEQIFNFCSSSNEKAEEFKEKDISQSIDFILKFSKCLQGNLSIIQDRIMLVIKAHPIYSSNSQIIYSTIIENLVIKEIDGVKVLQNVFLKSIIKDLQIWAKSICPQRLQS 275B942124926ECA q8mxz9_tetth TWI1 protein protein tetth Tetrahymena thermophila MSNKGLVQNNPRLRFLCNYYQVMLHNEKIYSYQFSVEGMEAAEVMKRTGEILKACINKLREVFTNKFLFQHSVLYSPVQLQANQMTEIGTVDTKTVSLKLLGEANNEREVQNIFGRLIKTVFAKIKLNKLGRKYFDPASRKEYNEYDLAVFSGYESSLEKYSFNQKLLNIDSCFKVQRTSNLLQALQQVKDRNDLSGFVGNTVITSYNGKFHRIESIEKDMSPNDAFEDRKGTKKTYMQYYKEAYNIGNIDPNQPLVKCVELKGKTKTPFTYYLIPSLCQVTGLTDQQRNDFNLMKKLAEVTKPKAGERIAQASKFIQRIKQDADKTLKDWSVEICEKPQTIDYKQLDAGNMEMQQGKKKINVATGSLDRDTQTQMFEQPSLNMWAIIYSDRDQKTFDSLLRTFQECLKFYNYPCKPPNCISVQSRGAQDWINALKNAPDNVQMAVFLLPGKKKAGMYYDEIKRYFTNVKPIPTQVILASTAMKDKGLRSVVNKLLMQICAKTGGVPWVMDNLPFQNLPTMVVGMDVFHNTPGKKESIFGFVSTVDRNFSKYYSHSHVLPTGQEITPFLQQVYEQALKEFKDSNGVYPQQIIIFRDGVGEGQFNAVKDIEMPQLKQACQKINGCENIKFTLIIVNKKVGAKFYQSQDGNVGSAENPPQGALIEDRVTKGVNDFFIVSQKTNQGTASPTHYTIIYNDMIDEALQKDNSPQYREFKRDLQVLAFKLCFLYYNWTGAIKTPSAVRYAHTLSNFVGDRYNPRKNDDTLIQAHPKYDKFRSLYFI 58D3FE686D6FE288 a4f2m3_tetth EMA1 protein protein tetth Tetrahymena thermophila MSNVNYKRPFQKMSVLIENASSEKTQSKAQDQDIQIINEPEKIKKIKLISDITKEYSKINPSSVQLSEKRNEKNQEKRNSFQINDVPKNTLFLNSGNRQDKFGEGQEDFMKFDEEIQISNTIVKEEIVVEEDQNNSNNRNKKKVFYEFNEPISPEQEELPVHENRLSILETIKNNSVIVISGNTGCGKSTQIPKFIACDDTSTKIIVTQPRRVAAIALAERVSQELKSNLGQRVGYQIGQDSNFNRNTQILYVTIGIFLQKLINDPDQFLREYTHIILDEVHERGIESDFGLIALKTFLSQHKNRNSIKLIVMSATLNKDIFLNYFSTTELNNLEKEIKEDQDCQEYYFDSSKFSEQLKITKQNDDYSNWGQQKAHQTWNDQDDHDDTYEKMQKMICRFNCKYESNQKNYVEIQVVQKYKIQTVWLEDLKDTQKLCNKNYFNARKPELNEDLYKIVAKEIKRVQFQEEPMLRSYKKGDILVFLPGYNEIVKLHSILDQIMMEFKQTNQSIYNQNKYKIIELHSCLGDINTKELFGRNQDYTKIILATNIAESSITIPNCFVVFDFCLTKEINYNPSSGIEKLELQWASKASITQRAGRTGRNCNGVNIRLVPKQFFMAEIKDYQTPEILRCPIEKVILKIKILDEGESDYQKIHLSDDDDVEQNNLIEEIIKEKQSEKSKKKRLNFSTDFDDIDYMKQFDTKPVFDGQDTAFLIAGPITKNKSKKQQLENSQNLRLFEDPVRVLQRAIEAPDDYSICQSMENLMSVGALKLKKIQYTDTQKEKIKVKVTKLGRFYNDMPCDLKLAKFICFGYFFDCLIECITIASIYSQRKSFFNPFYKISGNLKQIDQFTDLIFRYDQGQESDMILELKIFQEWENEFFQSQLEFIKLRKESKMQSRQIRQESEQNDFLSLDQSIPKILSEEQLKLPLSKQLELLKNSSNHPQNQDSSIAPTLSFQQYLPDRSSEEKPLKGIIDRYFIEEFKKKRASREELQWCEKHFCDPKVLREILSTREDLKQRIPKEAGSMYDIFEFKDLNDDSHLMKIKFCLAGAFNKYTLKSDIFDIKEMQKIRQYLLVEQMDPFSTIIIENTVYGFQSLSINDKDIIIKKIIDGVNHFIKTKQLFKELDDPYENVEALNNNFVSKIKLKYSSVYIEFKKFSKLPILLKQIMTFKKRYFNFIKLQDKDTNIKEISATIFLDKKIDFDQIFIKQQNNFKYAYEAKLNISNQKLNRLVSLESDSINLVTLGDYNRHKYLVASELMCRGHRVVAQGITVMPPNFPLFLAFAALIFGSQCYVNCDKDGKKYEGVNVDSVEIEIDHYMLDNDIKEINDIRRELRACLDEDSFFNGQANLQLDLWNKVKRKLLSRVMMRHVDSEKWEDIYSKEYLNGTFSVNVEEQNKQKVMKILNSIRTTNSSENCKTSECQNEEMDLEKNNNSLSIAQQNQKKRNVKLLFQNHKRKNNQEKITHASEKGFMNQILQTKKQESNLVTLSEQENINLLQEYFENYNIWKQMKMRELKRFFLLHTIDEPIITCKACYHQLLFQVPLFKANKLQAQLEKFMKLNFNGFTDNLYLKDIKIEQLLQEAEFNQDKTKYKTQQEKDLEQNADFIKELKYNFNQLNSQNSDQIYSFKYYACQNGHLIGFTQDDNQLYNQMHIPDCNQLLILYPDLEYKVFSGYEDFQEVKLTEDQQIEIQEHKALMLLELNEISCIICNCVMNDSEKKDEEIKFQDQNVTDWRSKLEKHMKTEAHKLNEEIFLKSTRSPNYFFTN F2D16D93DA2B105E giw1-twi1 5 7 unspecified role unspecified role bait bait c terminal binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 8 unspecified role unspecified role prey prey physical association physical association Fig 3B. A substantially higher amount of Twi1p was precipitated from the GIW1-HA strains than from the nontagged strains at all developmental stages tested, confirming that Twi1p and Giw1p are found in the same complex. twi1-giw1 5 7 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait Q8MXZ9:p.Asp526Asn mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain D N Wed May 02 16:16:44 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp526asn' Tue Jun 30 18:50:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp526Asn' Q8MXZ9:p.His745Gln mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain H Q Wed May 02 14:26:38 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'his745gln' Tue Jun 30 17:25:46 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.His745Gln' n terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Fig 3C, 6C, S5. Giw1p can interact with Twi1p in the absence of scnRNA in cell lysate. No detectable Giw1p was precipitated with (Slicer-dead mutants) Twi1p-D526N nor Twi1p-H745Q. twi1-giw1-1 2 8 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined paz domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain piwi domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro physical association physical association Fig 3D, 6D. Giw1p coprecipitated with the PAZ and the Piwi domains, but not with other domains of Twi1p. The amount of Giw1p coprecipitated with GST-Twi1p was greatly reduced when GST-Twi1p was preincubated with a guide and a noncleavable target RNA, indicating that the presence of double-stranded scnRNAs in Twi1p inhibits the Giw1p-Twi1p interaction. Double stranded scnRNAs. twi1-giw1-3 5 6 unspecified role unspecified role prey prey 9 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait n terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey physical association physical association Fig S3A. Small amounts of Ema1p and Wag1p were coimmunoprecipitated with Twi1p. twi1-giw1-4 1 8 unspecified role unspecified role bait bait n terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey physical association physical association Fig S3B. The 115 kDa protein was identified by mass spectrometry and named Giw1p (gentleman-in-waiting). twi1-giw1-5 4 8 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role prey prey in vitro In vitro physical association physical association Fig S3D. Giw1p was coimmunoprecipitated with Twi1p from EMA1 KO strains, in which the Twi1p-ncRNA interaction is impaired. Twi1p was immunoprecipitated using an anti-Twi1p antibody from wild-type (W) or EMA1 KO (Δ) strains at 4.5 hr and 9 hr post mixing and coimmunoprecipitated Giw1p was analyzed by Western blot using an anti-Giw1p antibody. twi1-giw1-6 3 8 unspecified role unspecified role prey prey paz domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined piwi domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait n3 domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association Fig S3E. Three of the six segments of Giw1p (N1, N3, and C2) were efficiently coprecipitated with PAZ and Piwi domains of Twi1p. Small amounts of the other three segments (N2, C1, and C3) were also coprecipitated with PAZ and Piwi domains. twi1-giw1-7 2 7 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 8 unspecified role unspecified role bait bait paz domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined piwi domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association Fig 3E. Three of the six segments of Giw1p (N1, N3, and C2) were efficiently coprecipitated with PAZ and Piwi domains of Twi1p. Small amounts of the other three segments (N2, C1, and C3) were also coprecipitated with PAZ and Piwi domains. you said not to add any domains - as they are all "positive" according to the paper. The only difference between Expt E2 and this one (E3) is that E2 uses "phosphoimager" for detection, while this one uses autoradiography- the paper regards them as distinct methods - but maybe they could be amalgamated? (especially as the interactions are the same)