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/ Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung. Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung. J. Biol. Chem. (0021-9258) 2006 imex curation Goswami SC., Yoon JH., Abramczyk BM., Pfeifer GP., Postel EH. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. posteleh@umdnj.edu 23-Feb-2010: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting autoradiography autoradiography Goswami SC., Yoon JH., Abramczyk BM., Pfeifer GP., Postel EH. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. posteleh@umdnj.edu Accepted 2009-Not a month!-4 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 orchard Only protein-protein interactions imex curation Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung. Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung. J. Biol. Chem. (0021-9258) 2006 imex curation Goswami SC., Yoon JH., Abramczyk BM., Pfeifer GP., Postel EH. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. posteleh@umdnj.edu 23-Feb-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 Goswami SC., Yoon JH., Abramczyk BM., Pfeifer GP., Postel EH. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. posteleh@umdnj.edu Accepted 2009-Not a month!-4 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 orchard Only protein-protein interactions imex curation Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung. Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung. J. Biol. Chem. (0021-9258) 2006 imex curation Goswami SC., Yoon JH., Abramczyk BM., Pfeifer GP., Postel EH. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. posteleh@umdnj.edu 23-Feb-2010: Contacted by JYOTI. orchard Only protein-protein interactions ecoli Escherichia coli (strain K12) anti bait coip anti bait coimmunoprecipitation anti bait coip experimental particp experimental participant identification experimental particp Goswami SC., Yoon JH., Abramczyk BM., Pfeifer GP., Postel EH. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. Immunoprecipitated (IP) cell extracts were extensively washed and diluted aliquots of the beads tested directly for UDG activity posteleh@umdnj.edu Accepted 2009-Not a month!-4 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 orchard Only protein-protein interactions imex curation ung_ecoli Uracil-DNA glycosylase ung b2580 JW2564 protein protein ecoli Escherichia coli (strain K12) MANELTWHDVLAEEKQQPYFLNTLQTVASERQSGVTIYPPQKDVFNAFRFTELGDVKVVILGQDPYHGPGQAHGLAFSVRPGIAIPPSLLNMYKELENTIPGFTRPNHGYLESWARQGVLLLNTVLTVRAGQAHSHASLGWETFTDKVISLINQHREGVVFLLWGSHAQKKGAIIDKQRHHVLKAPHPSPLSAHRGFFGCNHFVLANQWLEQRGETPIDWMPVLPAESE DIP protein P12295 original sequence version: 101 DIP protein P12295 original sequence version: null CD44F1E214FE74ED ndk_ecoli Nucleoside diphosphate kinase ndk b2518 Nucleoside-2-P kinase JW2502 protein protein ecoli Escherichia coli (strain K12) MAIERTFSIIKPNAVAKNVIGNIFARFEAAGFKIVGTKMLHLTVEQARGFYAEHDGKPFFDGLVEFMTSGPIVVSVLEGENAVQRHRDLLGATNPANALAGTLRADYADSLTENGTHGSDSVESAAREIAYFFGEGEVCPRTR 8C069C10043532EC 8C069C10043532EC ndk_ung 2 4 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) 5 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) physical association physical association Fig. 5 Binding assay using 2 μg of each purified recombinant GST, GST-Ung, and His-Ndk proteins ndk_ung-1 1 4 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 ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association To further probe for direct protein/protein interaction, we used a far Western protein-blotting technique Fig. 6 ndk_ung-2 3 5 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association Immunoprecipitated (IP) cell extracts were extensively washed and diluted aliquots of the beads tested directly for UDG activity Fig. 4