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/ Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells (1066-5099) 2005 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. orchard ecoli Escherichia coli (strain K12) 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 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr Accepted 2008-MAR-8 by ORCHARD. 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 orchard Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells (1066-5099) 2005 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. orchard ecoli Escherichia coli (strain K12) 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr Accepted 2008-MAR-8 by ORCHARD. 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 orchard Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells (1066-5099) 2005 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. orchard in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr Accepted 2008-MAR-8 by ORCHARD. 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 orchard Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells (1066-5099) 2005 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. orchard in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr Accepted 2008-MAR-8 by ORCHARD. 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 orchard Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells (1066-5099) 2005 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. orchard chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr Accepted 2008-MAR-8 by ORCHARD. 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 orchard Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells (1066-5099) 2005 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. orchard in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr Accepted 2008-MAR-8 by ORCHARD. 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 orchard Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells (1066-5099) 2005 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. orchard mouse-p19 Mus musculus embryonic teratocarcinoma cell line, epithelial-like, induced by retinoic acid p19 mouse embryonic teratocarcinoma cellline anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr Accepted 2008-MAR-8 by ORCHARD. 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 orchard Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells (1066-5099) 2005 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. orchard chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 fluorescence imaging fluorescence microscopy fluorescence imaging tag fluorescence tag visualisation by fluorescence tag fluorescence Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr Accepted 2008-MAR-8 by ORCHARD. 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 orchard Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells (1066-5099) 2005 Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. orchard mouse-p19 Mus musculus embryonic teratocarcinoma cell line, epithelial-like, induced by retinoic acid p19 mouse embryonic teratocarcinoma cellline fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Lee J., Rhee BK., Bae GY., Han YM., Kim J. Cancer - Interactions investigated in the context of cancer The Strategene webiste shows that the BacterioMatch Two-Hybrid system reporter strain used is derived from XL-1 Blue MRF, a strain of E. coli. jkim@sogang.ac.kr Accepted 2008-MAR-8 by ORCHARD. 01-April-2008: Contacted by JYOTI. A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 orchard po5f1_mouse POU domain, class 5, transcription factor 1 Pou5f1 Oct-3 Oct-4 Otf-3 Otf3 Octamer-binding transcription factor 3 NF-A3 Octamer-binding protein 3 Octamer-binding protein 4 protein protein mouse Mus musculus Mouse MAGHLASDFAFSPPPGGGDGSAGLEPGWVDPRTWLSFQGPPGGPGIGPGSEVLGISPCPPAYEFCGGMAYCGPQVGLGLVPQVGVETLQPEGQAGARVESNSEGTSSEPCADRPNAVKLEKVEPTPEESQDMKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSLKNMCKLRPLLEKWVEEADNNENLQEICKSETLVQARKRKRTSIENRVRWSLETMFLKCPKPSLQQITHIANQLGLEKDVVRVWFCNRRQKGKRSSIEYSQREEYEATGTPFPGGAVSFPLPPGPHFGTPGYGSPHFTTLYSVPFPEGEAFPSVPVTALGSPMHSN DIP protein P20263 original sequence version: 87 DIP protein P20263 original sequence version: 121 DIP protein P20263 original sequence version: 118 DIP protein P20263 original sequence version: 137 DIP protein P20263 original sequence version: 107 757E41DF52286714 ews_mouse RNA-binding protein EWS Ewsr1 Ews Ewsh protein protein mouse Mus musculus Mouse MASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDVSYTQAQTTATYGQTAYATSYGQPPTGYSTPTAPQAYSQPVQGYGTGTYDSTTATVTTTQASYAAQTAYGTQPAYPTYGQQPTATAPTRPQDGNKPAETSQPQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQPVTAPPSYPPTSYSSSQPTSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGSYSQAPSQYSQQSSSYGQQSSFRQDHPSSMGVYGQESGGFSGPGENRSLSGPDNRGRGRGGFDRGGMSRGGRGGGRGGLGAGERGGFNKPGGPMDEGPDLDLGLPIDPDEDSDNSAIYVQGLNDNVTLDDLADFFKQCGVVKMNKRTGQPMIHIYLDKETGKPKGDATVSYEDPPTAKAAVEWFDGKDFQGSKLKVSLARKKPPMNSMRGGMPPREGRGMPPPLRGGPGGPGGPGGPMGRMGGRGGDRGGFPPRGPRGSRGNPSGGGNVQHRAGDWQCPNPGCGNQNFAWRTECNQCKAPKPEGFLPPPFPPPGGDRGRGGPGGMRGGRGGLMDRGGPGGMFRGGRGGDRGGFRGGRGMDRGGFGGGRRGGPGGPPGPLMEQMGGRRGGRGGPGKMDKGEHRQERRDRPY 10C7F06A921668F3 pou5f1-ewsr1 2 10 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey physical association physical association we identified five interesting clones as the Oct-4-binding partners. In addition, through nucleotide sequence determination and comparison with GenBank and SwissProt databases, we found that one clone contained the cDNA sequence of EWS pou5f1-ewsr1-1 1 11 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait physical association physical association Fig. 1 C and D we found that only the expression of both pBT-Oct-4 and pTRG-EWS in bacterial reporter cell line allowed growth on +Kan/+Chl/+Tet LB plates containing 0.47 or 0.59 mM carbenicillin, indicating a physical interaction between Oct-4 and EWS pou5f1-ewsr1-2 3 10 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 ecoli Escherichia coli (strain K12) 11 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 direct interaction direct interaction Fig. 3 A As shown in Figure 3A, approximately 10% of input EWS protein was specifically retained on the Oct-4-conjugated Sepharose beads. Because EWS did not bind to GST alone (Fig. 3A, lane 2), the interaction was considered to be specific. pou5f1-ewsr1-3 6 11 unspecified role unspecified role prey prey mouse-p19 Mus musculus embryonic teratocarcinoma cell line, epithelial-like, induced by retinoic acid p19 mouse embryonic teratocarcinoma cellline 10 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 ecoli Escherichia coli (strain K12) physical association physical association Fig. 3 B GST-Oct-4 beads, but not control GST beads, efficiently retained endogenous EWS ewsr1-pou5f1 6 10 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 11 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 mouse-p19 Mus musculus embryonic teratocarcinoma cell line, epithelial-like, induced by retinoic acid p19 mouse embryonic teratocarcinoma cellline physical association physical association Fig. 3 B In the reciprocal experiment, the GST-EWS fusion protein was also able to interact with endogenous Oct-4 pou5f1-ewsr1-4 5 10 unspecified role unspecified role bait bait 11 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 3 C EWS was found to specifically coprecipitate with Oct-4 Q61545; P20263 transfection pou5f1-ewsr1-5 7 11 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait physical association physical association Fig. 3 D Antibody against Oct-4 (C-10, Santa Cruz Biotechnology), and not control serum, coprecipitated EWS ewsr1-pou5f1-1 7 10 unspecified role unspecified role prey prey 11 unspecified role unspecified role bait bait physical association physical association Fig. 3 D whereas anti-EWS (C-19, Santa Cruz Biotechnology) coprecipitated Oct-4 ewsr1-pou5f1-2 4 11 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 ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role prey prey 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 3 E bacterially produced GSTEWS protein interacts with recombinant Oct-4, suggesting that the Oct-4-EWS interaction does not require an adaptor protein. pou5f1-ewsr1-6 3 11 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 10 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 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 4 B EWS bound to GST-Oct-4 (POU) but did not interact with GST-Oct-4 (NTD) or GST-Oct-4 (CTD) ewsr1-pou5f1-3 4 10 unspecified role unspecified role bait bait 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 ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role prey prey 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 c-terminal 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 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro direct interaction direct interaction Fig. 5 This result suggests that EWS has at least three sites (amino acids 70 through 163, GRP I, and GRP II and III) that can bind to Oct-4 independently. This result indicates that EWS has at least three sites that can bind to Oct-4 independently. The UniProtKB entry for EWS Q61545 has only 655 residues whereas the diagram in Fig. 5 A shows the protein as being 656 aa in length. ewsr1-pou5f1-4 8 10 unspecified role unspecified role neutral component neutral component region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 11 unspecified role unspecified role neutral component neutral component region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization Fig. 6 A The overlay image indicated that Oct-4 and EWS in the nucleoplasm partially overlapped P20263; Q61545 transfection ewsr1-pou5f1-5 9 10 unspecified role unspecified role neutral component neutral component 11 unspecified role unspecified role neutral component neutral component colocalization colocalization Fig. 6 B the colocalization of the endogenously expressed Oct-4 and EWS proteins was also determined by indirect immunofluorescence studies using α-Oct-4 (C-10, Santa Cruz Biotechnology) or α-EWS (C-19, Santa Cruz Biotechnology) antibodies. As shown in Figure 6B, both proteins are exclusively localized to the nucleus in P19 cells,