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/ APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Mol. Cell. Biol. (0270-7306) 2007 imex curation Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk 07-Aug-2007: Contacted by JYOTI. Only protein-protein interactions human-a_549 Homo sapiens Caucasian lung carcinoma cell line a_549 Human Caucasian lung carcinoma fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk Accepted 2007-JUL-12 by JYOTI. 07-Aug-2007: Contacted by JYOTI. Mol. Cell. Biol. (0270-7306) 2007 Only protein-protein interactions imex curation APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Mol. Cell. Biol. (0270-7306) 2007 imex curation Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk 07-Aug-2007: Contacted by JYOTI. Only protein-protein interactions crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk Accepted 2007-JUL-12 by JYOTI. 07-Aug-2007: Contacted by JYOTI. Mol. Cell. Biol. (0270-7306) 2007 Only protein-protein interactions imex curation APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Mol. Cell. Biol. (0270-7306) 2007 imex curation Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk 07-Aug-2007: Contacted by JYOTI. Only protein-protein interactions 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 Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk Accepted 2007-JUL-12 by JYOTI. Mol. Cell. Biol. (0270-7306) 2007 Only protein-protein interactions imex curation APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Mol. Cell. Biol. (0270-7306) 2007 imex curation Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk 07-Aug-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting western blot western blot Immuno blot Immuno blot Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk Accepted 2007-JUL-12 by JYOTI. 07-Aug-2007: Contacted by JYOTI. Mol. Cell. Biol. (0270-7306) 2007 Only protein-protein interactions imex curation APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Mol. Cell. Biol. (0270-7306) 2007 imex curation Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk 07-Aug-2007: Contacted by JYOTI. Only protein-protein interactions human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk Accepted 2007-JUL-12 by JYOTI. 07-Aug-2007: Contacted by JYOTI. Mol. Cell. Biol. (0270-7306) 2007 Only protein-protein interactions imex curation APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Mol. Cell. Biol. (0270-7306) 2007 imex curation Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk 07-Aug-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro filter binding filter binding Filter overlay assay Filter overlay assay antibody detection identification by antibody antibody detection Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk Accepted 2007-JUL-12 by JYOTI. 07-Aug-2007: Contacted by JYOTI. Mol. Cell. Biol. (0270-7306) 2007 Only protein-protein interactions imex curation APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks. Mol. Cell. Biol. (0270-7306) 2007 imex curation Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk 07-Aug-2007: Contacted by JYOTI. Only protein-protein interactions human-a_549 Homo sapiens Caucasian lung carcinoma cell line a_549 Human Caucasian lung carcinoma anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Iles N., Rulten S., El-Khamisy SF., Caldecott KW. k.w.caldecott@sussex.ac.uk Accepted 2007-JUL-12 by JYOTI. 07-Aug-2007: Contacted by JYOTI. Mol. Cell. Biol. (0270-7306) 2007 Only protein-protein interactions imex curation xrcc1_human DNA repair protein XRCC1 XRCC1 X-ray repair cross-complementing protein 1 protein protein human Homo sapiens Human MPEIRLRHVVSCSSQDSTHCAENLLKADTYRKWRAAKAGEKTISVVLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGAGEQDYEVLLVTSSFMSPSESRSGSNPNRVRMFGPDKLVRAAAEKRWDRVKIVCSQPYSKDSPFGLSFVRFHSPPDKDEAEAPSQKVTVTKLGQFRVKEEDESANSLRPGALFFSRINKTSPVTASDPAGPSYAAATLQASSAASSASPVSRAIGSTSKPQESPKGKRKLDLNQEEKKTPSKPPAQLSPSVPKRPKLPAPTRTPATAPVPARAQGAVTGKPRGEGTEPRRPRAGPEELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTRDSTHLICAFANTPKYSQVLGLGGRIVRKEWVLDCHRMRRRLPSRRYLMAGPGSSSEEDEASHSGGSGDEAPKLPQKQPQTKTKPTQAAGPSSPQKPPTPEETKAASPVLQEDIDIEGVQSEGQDNGAEDSGDTEDELRRVAEQKEHRLPPGQEENGEDPYAGSTDENTDSEEHQEPPDLPVPELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDNMSDRVQFVITAQEWDPSFEEALMDNPSLAFVRPRWIYSCNEKQKLLPHQLYGVVPQA DIP protein P18887 original sequence version: 120 DIP protein P18887 original sequence version: 161 30DB3321234DBFC2 aplf_human Aprataxin and PNK-like factor APLF C2orf13 PALF XIP1 Apurinic-apyrimidinic endonuclease APLF PNK and APTX-like FHA domain-containing protein XRCC1-interacting protein 1 protein protein human Homo sapiens Human MSGGFELQPRDGGPRVALAPGETVIGRGPLLGITDKRVSRRHAILEVAGGQLRIKPIHTNPCFYQSSEKSQLLPLKPNLWCYLNPGDSFSLLVDKYIFRILSIPSEVEMQCTLRNSQVLDEDNILNETPKSPVINLPHETTGASQLEGSTEIAKTQMTPTNSVSFLGENRDCNKQQPILAERKRILPTWMLAEHLSDQNLSVPAISGGNVIQGSGKEEICKDKSQLNTTQQGRRQLISSGSSENTSAEQDTGEECKNTDQEESTISSKEMPQSFSAITLSNTEMNNIKTNAQRNKLPIEELGKVSKHKIATKRTPHKEDEAMSCSENCSSAQGDSLQDESQGSHSESSSNPSNPETLHAKATDSVLQGSEGNKVKRTSCMYGANCYRKNPVHFQHFSHPGDSDYGGVQIVGQDETDDRPECPYGPSCYRKNPQHKIEYRHNTLPVRNVLDEDNDNVGQPNEYDLNDSFLDDEEEDYEPTDEDSDWEPGKEDEEKEDVEELLKEAKRFMKRK DIP protein Q8IW19 original sequence version: 82 DIP protein Q8IW19 original sequence version: 58 CBBF0096843298DA xrcc4_human DNA repair protein XRCC4 X-ray repair cross-complementing protein 4 XRCC4 protein protein human Homo sapiens Human MERKISRIHLVSEPSITHFLQVSWEKTLESGFVITLTDGHSAWTGTVSESEISQEADDMAMEKGKYVGELRKALLSGAGPADVYTFNFSKESCYFFFEKNLKDVSFRLGSFNLEKVENPAEVIRELICYCLDTIAENQAKNEHLQKENERLLRDWNDVQGRFEKCVSAKEALETDLYKRFILVLNEKKTKIRSLHNKLLNAAQEREKDIKQEGETAICSEMTADRDPVYDESTDEESENQTDLSGLASAAVSKDDSIISSLDVTDIAPSRKRRQRMQRNLGTEPKMAPQENQLQEKENSRPDSSLPETSKKEHISAENMSLETLRNSSPEDLFDEI DIP protein Q13426 original sequence version: 140 DIP protein Q13426-1 original sequence version: 116 BE5FB99153479A4E apfl-xrcc1-1 3 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association APLF interacts with the DNA strand break repair proteins XRCC1 and XRCC4. Indeed, APLF interacted with both of these proteins in yeast two-hybrid analyses 2A apfl-xrcc4-1 3 10 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association APLF interacts with the DNA strand break repair proteins XRCC1 and XRCC4. Indeed, APLF interacted with both of these proteins in yeast two-hybrid analyses 2A apfl-xrcc1-2 5 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association In addition, endogenous XRCC1 was immunoprecipitated from Myc-APLF-transfected HeLa cell extracts by anti-Myc antibodies but was not immunoprecipitated by control anti-FLAG antibodies (Fig. 2B, upper panel) or by anti-Myc antibodies from untransfected cells (Fig. 2B, lower panel). 2B apfl-xrcc1-3 7 9 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait ser-485 (possible) opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain thr-488 (possible) opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain physical association physical association More importantly, immunoprecipitation of endogenous XRCC1 from the human A549 cell extract, which we detected using both standard anti-XRCC1 polyclonal antibodies and antibodies specific for CK2-phosphorylated XRCC1(pS485/pT488) (21, 22), corecovered endogenous APLF, 2C apfl-xrcc1-4 6 8 unspecified role unspecified role prey prey ser-485 (possible) opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain thr-488 (possible) opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain ser-518 (possible) opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain thr-519 (possible) opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain thr-523 (possible) opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt 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 ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role bait bait fha 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 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 Whereas both His-APLF and His-APLF1-166 preferentially interacted with CK2-phosphorylated XRCC1-His (Fig. 3C, top left and middle panels), neither protein bound to control slots blotted with CK2 reaction products lacking XRCC1-His (Fig. 3C, bottom left panel). 3B. 3C Ser-519 and Ser-523 described in the paper, actually appear to be threonine residues. APLF interacts with the phosphorylated form of XRCC1. The phosphorylated residues were first identified by incubating the XRCC1 with CK2 under the same conditions as used in this experiment and demonstrating site specific phosphorylation by use of the appropriate antibodies. apfl-xrcc1-5 4 9 unspecified role unspecified role prey prey fha 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 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) 8 unspecified role unspecified role bait bait ser-485 (possible) opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain thr-488 (possible) opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain ser-518 (possible) opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain thr-519 (possible) opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain thr-523 (possible) opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt 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 ecoli Escherichia coli (strain K12) physical association physical association Similar results were observed by far-Western blotting, in which the CK2-phosphorylated XRCC1-His protein probe detected His-APLF and His-APLF1-166, but not His-APLF360-511, proteins present on renatured Western blots (Fig. 3D). 3D Ser-519 and Ser-523 described in the paper, actually appear to be threonine residues. APLF interacts with the phosphorylated form of XRCC1. The phosphorylated residues were first identified by incubating the XRCC1 with CK2 under the same conditions as used in this experiment and demonstrating site specific phosphorylation by use of the appropriate antibodies. apfl-xrcc1-6 1 8 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component physical association physical association More significantly, APLF colocalized and accumulated with XRCC1 at these sites, confirming that APLF is a component of the response to UVA-induced cellular damage. 4A 0.35 uJ/um2 UVA apfl-xrcc1-7 2 9 unspecified role unspecified role neutral component neutral component region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Q8IW19:p.Arg27Ala mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain R A Wed May 02 18:50:18 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg27ala' Tue Jun 30 18:00:13 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg27Ala' 8 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 physical association physical association The fraction of cells with largely nuclear YFP-APLF transiently increased after H2O2 treatment in an RFP-XRCC1-dependent manner (Fig. 4C, middle and right panels). 4C 10 mM H2O2 for 10 min Arg27Ala mutant of APLF remains in the cytoplasm after hydrogen peroxide treatment.