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/ Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Dev. Biol. (0012-1606) 2004 Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu 22-Jan-2008: Contacted by JYOTI. 13-Nov-2007: Contacted by JYOTI. orchard yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu Accepted 2007-DEC-21 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Dev. Biol. (0012-1606) 2004 orchard Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Dev. Biol. (0012-1606) 2004 Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu 22-Jan-2008: Contacted by JYOTI. 13-Nov-2007: Contacted by JYOTI. orchard drome Drosophila melanogaster (Fruit fly) Fruit fly fluorescence imaging fluorescence microscopy fluorescence imaging predetermined predetermined participant predetermined Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu Accepted 2007-DEC-21 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Dev. Biol. (0012-1606) 2004 orchard Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Dev. Biol. (0012-1606) 2004 Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu 22-Jan-2008: Contacted by JYOTI. 13-Nov-2007: Contacted by JYOTI. orchard yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu Accepted 2007-DEC-21 by ORCHARD. Dev. Biol. (0012-1606) 2004 orchard Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Dev. Biol. (0012-1606) 2004 Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu 22-Jan-2008: Contacted by JYOTI. 13-Nov-2007: Contacted by JYOTI. orchard drome-schneider_2 Drosophila melanogaster late embryo cells Fruit fly schneider_2 Established from the late embryo of Drosophila melanogaster Mixture of diploid and tetraploid. Originally had some XY cells now predominantly XX. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu Accepted 2007-DEC-21 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Dev. Biol. (0012-1606) 2004 orchard Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Dev. Biol. (0012-1606) 2004 Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu 22-Jan-2008: Contacted by JYOTI. 13-Nov-2007: Contacted by JYOTI. orchard drome-schneider_2 Drosophila melanogaster late embryo cells Fruit fly schneider_2 Established from the late embryo of Drosophila melanogaster Mixture of diploid and tetraploid. Originally had some XY cells now predominantly XX. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu Accepted 2007-DEC-21 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Dev. Biol. (0012-1606) 2004 orchard Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Dev. Biol. (0012-1606) 2004 Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu 22-Jan-2008: Contacted by JYOTI. 13-Nov-2007: Contacted by JYOTI. orchard in vitro In vitro pull down pull down predetermined predetermined participant predetermined Wang G., Jiang J. Various N-terminal fragments of Ci and CORD were fused in frame with the Gal4 activation domain. The resulting Ci/Gal4 fusion constructs were transformed into yeast with a Cos2/LexA DNA binding domain fusion construct. Interactions were determined by a liquid assay. The relative strength of protein - protein interaction is reflected by the beta - galactosidase activity. jin.jiang@utsouthwestern.edu Accepted 2007-DEC-21 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Dev. Biol. (0012-1606) 2004 orchard ci_drome Transcriptional activator cubitus interruptus ci ci-D CG2125 ci form of 155 kDa ci full-length protein protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MDAYALPTYFPLAYSELQFLASRRAAAVAAAATVLPGSPCINQHHPTDVSSSVTVPSIIPTGGTSDSIKTSIQPQICNENTLLGNAGHQHNHQPQHVHNINVTGQPHDFHPAYRIPGYMEQLYSLQRTNSASSFHDPYVNCASAFHLAGLGLGSADFLGSRGLSSLGELHNAAVAAAAAGSLASTDFHFSVDGNRRLGSPRPPGGSIRASISRKRALSSSPYSDSFDINSMIRFSPNSLATIMNGSRGSSAASGSYGHISATALNPMSHVHSTRLQQIQAHLLRASAGLLNPMTPQQVAASGFSIGHMPTSASLRVNDVHPNLSDSHIQITTSPTVTKDVSQVPAAAFSLKNLDDAREKKGPFKDVVPEQPSSTSGGVAQVEADSASSQLSDRCYNNVVNNITGIPGDVKVNSRLDEYINCGSISIPSNEYDCANADTTDIKDEPGDFIETNCHWRSCRIEFITQDELVKHINNDHIQTNKKAFVCRWEDCTRGEKPFKAQYMLVVHMRRHTGEKPHKCTFEGCFKAYSRLENLKTHLRSHTGEKPYTCEYPGCSKAFSNASDRAKHQNRTHSNEKPYICKAPGCTKRYTDPSSLRKHVKTVHGAEFYANKKHKGLPLNDANSRLQQNNSRHNLQEHNIDSSPCSEDSHLGKMLGTSSPSIKSESDISSSNHHLVNGVRASDSLLTYSPDDLAENLNLDDGWNCDDDVDVADLPIVLRAMVNIGNGNASASTIGGSVLARQRFRGRLQTKGINSSTIMLCNIPESNRTFGISELNQRITELKMEPGTDAEIKIPKLPNTTIGGYTEDPLQNQTSFRNTVSNKQGTVSGSIQGQFRRDSQNSTASTYYGSMQSRRSSQSSQVSSIPTMRPNPSCNSTASFYDPISPGCSRRSSQMSNGANCNSFTSTSGLPVLNKESNKSLNACINKPNIGVQGVGIYNSSLPPPPSSHLIATNLKRLQRKDSEYHNFTSGRFSVPSYMHSLHIKNNKPVGENEFDKAIASNARRQTDPVPNINLDPLTNISRFSTTPHSFDINVGKTNNIASSINKDNLRKDLFTVSIKADMAMTSDQHPNERINLDEVEELILPDEMLQYLNLVKDDTNHLEKEHQAVPVGSNVSETIASNHYREQSNIYYTNKQILTPPSNVDIQPNTTKFTVQDKFAMTAVGGSFSQRELSTLAVPNEHGHAKCESFHHQSQKYMNTDIGSKQQSALPSAHQRQTEKSNYNQIIDSSMTSLPELNVDSIYPRNETENIFKVHGDHDNEIQCGIISQSQMSPSTNLNNDGQFSTVNMQPITTSKLFPPEPQKIVCDTQASNTSVMHLDTYQRTLEYVQSCQNWMETNNTSTNQIQSLPGMPVNNTLFPDVSSSTHPYHGTNMVINDMTTSLTSLLEENRYLQMMQ DIP protein P19538 original sequence version: 136 DIP protein P19538 original sequence version: 27 C3232A71EF9B8004 cos_drome Kinesin-like protein costa cos cos2 costal-2 Kinesin-like protein costal2 CG1708 protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MEIPIQVAVRIFPHRELKDLLRSFGPTEPKKDAQAVDEGADSKDSEAQVPAAEKDNPSISETDPNGNAEQDSAADSKTIPDANGNDSGQKDYPDSAYCVQAIPISASALGLPSALPGGDPMDSIAAGLIQVGPHTVPVTHALPSSSSQEQVYHQTVFPLITLFLEGFDASVVTYGQRGQGKSYTLYGNVQDPTLTDSTEGVVQLCVRDIFSHISLHPERTYAINVGFVEICGGDVCDLLGMGNIHCTNVDAVFHWLQVGLSARQSLPAHTLFTLTLEQQWVSKEGLLQHRLSTASFSDLCGTERCGDQPPGRPLDAGLCMLEQVISTLTDPGLMYGVNGNIPYGQTTLTTLLKDSFGGRAQTLVILCVSPLEEHLPETLGNLQFAFKVQCVRNFVIMNTYSDDNTMIVQPAEPVPESNSSAGPLSQAGPGDNFGLQFAASQWSKLVTNAEGLFSKLIDSKLITEVEKEQIEEWLFLKQECEECLSSTEAMRQQKQLVPILEAEEPEDVNSEAANSESPNSDNENDTDNESHRPDLDDKIESLMEEFRDKTDALILEKHAEYLSKHPKAVMQSQDREIEAQPPEENGDDRKVSIGSRRRSVQPGASLSTAELAMLNRVASQQPPPPIDPESVVDPLESSSGEGIRQAALAAAAATAPIEQLQKKLRKLVAEIEGKQRQLREIEETIQVKQNIIAELVKNSDTRSHAKQRFHKKRAKLEAECDKAKKQLGKALVQGRDQSEIERWTTIIGHLERRLEDLSSMKHIAGESGQKVKKLQQSVGESRKQADDLQKKLRKECKLRCQMEAELAKLRESRETGKELVKAQGSPEQQGRQLKAVQARITHLNHILREKSDNLEEQPGPEQQETLRHEIRNLRGTRDLLLEERCHLDRKLKRDKVLTQKEERKLLECDEAIEAIDAAIEFKNEMITGHRSIDTSDRIQREKGEQMLMARLNRLSTEEMRTLLYKYFTKVIDLRDSSRKLELQLVQLERERDAWEWKERVLSNAVRQARLEGERNAVLLQRQHEMKLTLMLRHMAEETSASSASYGERALAPACVAPPVQASSDFDYDHFYKGGGNPSKALIKAPKPMPTGSALDKYKDKEQRSGRNIFAKFHVLTRYASAAAAGSSGSTAEESTALIESTTTATATTTSTTTTGAVGKVKDKALVSFRPEQLKRLMPAPTATKVTRQKNKIIIQDASRRN DIP protein O16844 original sequence version: 88 DIP protein O16844 original sequence version: 100 DIP protein O16844 original sequence version: 121 6DC898F8B4C8F20F fused_drome Serine/threonine-protein kinase fused fu CG6551 protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MNRYAVSSLVGQGSFGCVYKATRKDDSKVVAIKVISKRGRATKELKNLRRECDIQARLKHPHVIEMIESFESKTDLFVVTEFALMDLHRYLSYNGAMGEEPARRVTGHLVSALYYLHSNRILHRDLKPQNVLLDKNMHAKLCDFGLARNMTLGTHVLTSIKGTPLYMAPELLAEQPYDHHADMWSLGCIAYESMAGQPPFCASSILHLVKMIKHEDVKWPSTLTSECRSFLQGLLEKDPGLRISWTQLLCHPFVEGRIFIAETQAEAAKESPFTNPEAKVKSSKQSDPEVGDLDEALAALDFGESRQENLTTSRDSINAIAPSDVEHLETDVEDNMQRVVVPFADLSYRDLSGVRAMPMVHQPVINSHTCFVSGNSNMILNHMNDNFDFQASLRGGGVAAKPIVAPTVRQSRSKDLEKRKLSQNLDNFSVRLGHSVDHEAQRKATEIATQEKHNQENKPPAEAISYANSQPPQQQPQQLKHSMHSTNEEKLSSDNTPPCLLPGWDSCDESQSPPIENDEWLAFLNRSVQELLDGELDSLKQHNLVSIIVAPLRNSKAIPRVLKSVAQLLSLPFVLVDPVLIVDLELIRNVYVDVKLVPNLMYACKLLLSHKQLSDSAASAPLTTGSLSRTLRSIPELTVEELETACSLYELVCHLVHLQQQFLTQFCDAVAILAASDLFLNFLTHDFRQSDSDAASVRLAGCMLALMGCVLRELPENAELVERIVFNPRLNFVSLLQSRHHLLRQRSCQLLRLLARFSLRGVQRIWNGELRFALQQLSEHHSYPALRGEAAQTLDEISHFTFFVT DIP protein P23647 original sequence version: 121 DIP protein P23647 original sequence version: 147 A7804E2749B79D0C ci-cos-1 3 8 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1541232]The shifted ranges (143-750) couldn't be applied as the new full feature sequence is different from the previous one. [EBI-1541232]143-750 [EBI-1541232]O16844,1 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 7 unspecified role unspecified role bait bait c terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 17 12 13 18 16 14 physical association physical association 5 Cos2 sequence involved in CDN binding is dispersed. By contrast, the Cos2 sequence that mediates CORD interaction can be narrowed down to two small discrete regions as Cos2 fragments both from aa 143 to aa 343 and from aa 343 to aa 447 interact with CiC fu-cos-1 3 8 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1394153]The shifted ranges (547-750) couldn't be applied as the new full feature sequence is different from the previous one. [EBI-1394153]547-750 [EBI-1394153]O16844,1 9 unspecified role unspecified role prey prey physical association physical association 5 Cos2 fragment containing sequence from aa 547 to aa 750 (Cos2 547�750) is sufficient to bind Fu. ci-fu-1 3 9 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait c terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 5 we tested whether LexA�CiN or LexA�CiC interacts with the Fu-GAD fusion protein in yeast. As shown in Fig. 5C, CiC but not CiN binds Fu ci-cos-2 1 8 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey c terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 3A, 3B Ci fragment from aa 1 to aa 440 interacts with Cos2 in yeast, albeit with an affinity much lower than that of CORD/Cos2 interaction (Fig. 3B). Deletion from either end (Ci 212 440 or Ci 1 346) greatly reduces but not completely abolishes Cos2 binding, suggesting that the N-terminal Ci sequence required for Cos2 binding is dispersed. A dispersed N terminal and the CORD domain of Ci are involved in interaction with Cos. ci-fu-2 5 7 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role prey prey region 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 3C Flag-Cos2 was coprecipitated with HA-Ci76 but not with HA-Ci76ΔCDN whereas equal amounts of Flag-Su(fu) were coprecipitated with both HA-Ci76 and HA-CiΔCDN. Hence, deletion of the Ci sequence from aa 346 to aa 440 compromises Cos2 binding to the N-terminal region of Ci, but does not significantly perturb Ci/Su(fu) interaction. ci-cos-3 4 8 unspecified role unspecified role prey prey region 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 bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 3C Flag-Cos2 was coprecipitated with HA-Ci76 but not with HA-Ci76ΔCDN whereas equal amounts of Flag-Su(fu) were coprecipitated with both HA-Ci76 and HA-CiΔCDN. Hence, deletion of the Ci sequence from aa 346 to aa 440 compromises Cos2 binding to the N-terminal region of Ci, but does not significantly perturb Ci/Su(fu) interaction. ci-cos-4 6 7 unspecified role unspecified role bait bait region 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) 8 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 9 inhibitor inhibitor unspecified role unspecified role 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 association association 6B Recombinant GST-Ci76 fusion proteins were purified using Glutathione Sepharose 4B beads from bacterial extracts, and were incubated with in vitro translated 35S-labeled Su(fu) or Cos2 without or with increasing amounts of the corresponding competing protein. As shown in Fig. 6A, less Su(fu) was pulled down by GST-Ci76 in the presence of competing Cos2. Reciprocally, increasing amounts of Su(fu) decreased the amounts of Cos2 bound to GST-Ci76 (Fig. 6B). ci-cos-5 6 7 unspecified role unspecified role bait bait region 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) 8 inhibitor inhibitor 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 9 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 association association 6A Recombinant GST-Ci76 fusion proteins were purified using Glutathione Sepharose 4B beads from bacterial extracts, and were incubated with in vitro translated 35S-labeled Su(fu) or Cos2 without or with increasing amounts of the corresponding competing protein. As shown in Fig. 6A, less Su(fu) was pulled down by GST-Ci76 in the presence of competing Cos2. Reciprocally, increasing amounts of Su(fu) decreased the amounts of Cos2 bound to GST-Ci76 (Fig. 6B). ci-fu-3 6 7 unspecified role unspecified role bait bait region 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) 9 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 6A Recombinant GST-Ci76 fusion proteins were purified using Glutathione Sepharose 4B beads from bacterial extracts, and were incubated with in vitro translated 35S-labeled Su(fu) or Cos2 without or with increasing amounts of the corresponding competing protein. As shown in Fig. 6A, less Su(fu) was pulled down by GST-Ci76 in the presence of competing Cos2. Reciprocally, increasing amounts of Su(fu) decreased the amounts of Cos2 bound to GST-Ci76 (Fig. 6B). ci-cos-6 6 8 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 7 unspecified role unspecified role bait bait region 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 6B Recombinant GST-Ci76 fusion proteins were purified using Glutathione Sepharose 4B beads from bacterial extracts, and were incubated with in vitro translated 35S-labeled Su(fu) or Cos2 without or with increasing amounts of the corresponding competing protein. As shown in Fig. 6A, less Su(fu) was pulled down by GST-Ci76 in the presence of competing Cos2. Reciprocally, increasing amounts of Su(fu) decreased the amounts of Cos2 bound to GST-Ci76 (Fig. 6B). ci-cos-7 4 7 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 inhibitor inhibitor unspecified role unspecified role region 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 9 unspecified role unspecified role prey prey region 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 association association 6C, 6D Cell extracts were immunoprecipitated with an anti-HA antibody, followed by Western blot analysis with either anti-Su(fu) or anti-Flag antibody. As shown in Fig. 6C, Su(fu) was coimmunoprecipitated with CiΔCORD in the absence of Flag-Cos2 (lane1); however, a diminishing amount of Su(fu) was coprecipitated with CiΔCORD when Flag-Cos2 was coexpressed (lane 2), suggesting that binding of Cos2 to CiΔCORD interferes with Su(fu)/CiΔCORD interaction. ci-fu-4 4 9 unspecified role unspecified role prey prey region 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 bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 6C, 6D Cell extracts were immunoprecipitated with an anti-HA antibody, followed by Western blot analysis with either anti-Su(fu) or anti-Flag antibody. As shown in Fig. 6C, Su(fu) was coimmunoprecipitated with CiΔCORD in the absence of Flag-Cos2 (lane1); however, a diminishing amount of Su(fu) was coprecipitated with CiΔCORD when Flag-Cos2 was coexpressed (lane 2), suggesting that binding of Cos2 to CiΔCORD interferes with Su(fu)/CiΔCORD interaction. ci-cos-11 2 7 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role neutral component neutral component colocalization colocalization 2 N-terminal region of Ci from aa 346 to aa 440, which we named CDN (Cos2 Responsive Domain in the N-terminal region of Ci), and CDN can mediate cytoplasmic retention by Cos2.