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/ http://www.ebi.ac.uk/ lee-2005b-3 Stimulation of Oct-4 activity by Ewing's sarcoma protein. 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 A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 lee-2005b-4 Stimulation of Oct-4 activity by Ewing's sarcoma protein. 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 A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 lee-2005b-6 Stimulation of Oct-4 activity by Ewing's sarcoma protein. 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 A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 lee-2005b-7 Stimulation of Oct-4 activity by Ewing's sarcoma protein. 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 A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 lee-2005b-1 Stimulation of Oct-4 activity by Ewing's sarcoma protein. 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. jkim@sogang.ac.kr A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 lee-2005b-2 Stimulation of Oct-4 activity by Ewing's sarcoma protein. 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 A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 lee-2005b-5 Stimulation of Oct-4 activity by Ewing's sarcoma protein. 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 A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 lee-2005b-8 Stimulation of Oct-4 activity by Ewing's sarcoma protein. 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 A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 lee-2005b-9 Stimulation of Oct-4 activity by Ewing's sarcoma protein. 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 A mouse ES cell-derived cDNA library was used. Stem Cells (1066-5099) 2005 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 ews_mouse RNA-binding protein EWS Ewsr1 Ews Ewsh protein protein mouse Mus musculus Mouse MASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDVSYTQAQTTATYGQTAYATSYGQPPTGYSTPTAPQAYSQPVQGYGTGTYDSTTATVTTTQASYAAQTAYGTQPAYPTYGQQPTATAPTRPQDGNKPAETSQPQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQPVTAPPSYPPTSYSSSQPTSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGSYSQAPSQYSQQSSSYGQQSSFRQDHPSSMGVYGQESGGFSGPGENRSLSGPDNRGRGRGGFDRGGMSRGGRGGGRGGLGAGERGGFNKPGGPMDEGPDLDLGLPIDPDEDSDNSAIYVQGLNDNVTLDDLADFFKQCGVVKMNKRTGQPMIHIYLDKETGKPKGDATVSYEDPPTAKAAVEWFDGKDFQGSKLKVSLARKKPPMNSMRGGMPPREGRGMPPPLRGGPGGPGGPGGPMGRMGGRGGDRGGFPPRGPRGSRGNPSGGGNVQHRAGDWQCPNPGCGNQNFAWRTECNQCKAPKPEGFLPPPFPPPGGDRGRGGPGGMRGGRGGLMDRGGPGGMFRGGRGGDRGGFRGGRGMDRGGFGGGRRGGPGGPPGPLMEQMGGRRGGRGGPGKMDKGEHRQERRDRPY pou5f1-ewsr1-2 GST-Oct-4 pulls down EWS in vitro 720739 N/A 720748 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 false ecoli Escherichia coli (strain K12) N/A 720749 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 false in vitro In vitro direct interaction direct interaction false false false Fig. 3 A pou5f1-ewsr1-6 GST-Oct-4 (POU domain) pulls down EWS in vitro 720739 N/A 720749 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 false in vitro In vitro N/A 720748 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 false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig. 4 B pou5f1-ewsr1-3 GST-Oct-4 pulls down EWS from P19 EC cell lysate 720740 N/A 720749 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 N/A 720748 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 false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig. 3 B ewsr1-pou5f1 GST-EwS pulls down Oct-4 from P19 EC cell lysate 720740 N/A 720748 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) N/A 720749 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 false mouse-p19 Mus musculus embryonic teratocarcinoma cell line, epithelial-like, induced by retinoic acid p19 mouse embryonic teratocarcinoma cellline physical association physical association false false false Fig. 3 B pou5f1-ewsr1-5 Oct-4 coimmunopreciptiated EWS in P19 lysates 720741 N/A 720749 unspecified role unspecified role prey prey N/A 720748 unspecified role unspecified role bait bait physical association physical association false false false Fig. 3 D ewsr1-pou5f1-1 Oct-4 coimmunoprecipitated with EWS in P19 lysates 720741 N/A 720748 unspecified role unspecified role prey prey N/A 720749 unspecified role unspecified role bait bait physical association physical association false false false Fig. 3 D ewsr1-pou5f1-2 GST-EWS pulls down Oct-4 in vitro 720742 N/A 720749 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 false ecoli Escherichia coli (strain K12) N/A 720748 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 false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Fig. 3 E ewsr1-pou5f1-3 GST-EWS constructs pull down Oct-4 in vitro 720742 N/A 720748 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 false ecoli Escherichia coli (strain K12) N/A 720749 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 false 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 false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false in vitro In vitro direct interaction direct interaction false false false Fig. 5 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. pou5f1-ewsr1 Oct-4 and EWS interact in a bacterial two-hybrid assay 720743 N/A 720748 unspecified role unspecified role bait bait N/A 720749 unspecified role unspecified role prey prey physical association physical association false false false pou5f1-ewsr1-1 Oct-4 and EWS interact in a directed bacterial two-hybrid assay 720744 N/A 720749 unspecified role unspecified role prey prey N/A 720748 unspecified role unspecified role bait bait physical association physical association false false false Fig. 1 C and D pou5f1-ewsr1-4 Oct-4 coimmunoprecipitates EWS in Cos-7 lysates 720745 N/A 720748 unspecified role unspecified role bait bait N/A 720749 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 false physical association physical association false false false 3 C Q61545; P20263 transfection ewsr1-pou5f1-4 GFP-EWS and RFP-Oct-4 colocalize in COS-7 cells 720746 N/A 720748 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 false N/A 720749 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 false colocalization colocalization false false false Fig. 6 A P20263; Q61545 transfection ewsr1-pou5f1-5 Endogenous EWS and Oct-4 colocalize in the nucleus of P19 cells 720747 N/A 720748 unspecified role unspecified role neutral component neutral component N/A 720749 unspecified role unspecified role neutral component neutral component colocalization colocalization false false false Fig. 6 B