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/ chattopadhyay-2006-4 in vitro In vitro crosslink cross-linking study crosslink weight by comassie molecular weight estimation by coomasie staining weight by comassie Chattopadhyay K., Bhatia S., Fiser A., Almo SC., Nathenson SG. Crosslinker:BS3 (bis(sulfosuccinimidyl)suberate), in presence of 2-mercaptoethanol, thus reducing disulfide linkages. nathenso@aecom.yu.edu,almo@aecom.yu.edu Journal of immunology (Baltimore, Md. : 1950) 2006 Only protein-protein interactions imex curation imex curation chattopadhyay-2006-5 in vitro In vitro facs fluorescence-activated cell sorting FACS Flow cytometry facs antibody detection identification by antibody antibody detection Chattopadhyay K., Bhatia S., Fiser A., Almo SC., Nathenson SG. native PAGE - non-denaturing. nathenso@aecom.yu.edu,almo@aecom.yu.edu Journal of immunology (Baltimore, Md. : 1950) 2006 Only protein-protein interactions imex curation imex curation chattopadhyay-2006-3 in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting western blot western blot Immuno blot Immuno blot Chattopadhyay K., Bhatia S., Fiser A., Almo SC., Nathenson SG. native PAGE - non-denaturing. nathenso@aecom.yu.edu,almo@aecom.yu.edu Journal of immunology (Baltimore, Md. : 1950) 2006 Only protein-protein interactions imex curation imex curation chattopadhyay-2006-1 in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Chattopadhyay K., Bhatia S., Fiser A., Almo SC., Nathenson SG. nathenso@aecom.yu.edu,almo@aecom.yu.edu Journal of immunology (Baltimore, Md. : 1950) 2006 Only protein-protein interactions imex curation imex curation chattopadhyay-2006-2 in vitro In vitro comig non denat gel comigration in non denaturing gel electrophoresis comig non denat gel weight by comassie molecular weight estimation by coomasie staining weight by comassie Chattopadhyay K., Bhatia S., Fiser A., Almo SC., Nathenson SG. nathenso@aecom.yu.edu,almo@aecom.yu.edu Journal of immunology (Baltimore, Md. : 1950) 2006 Only protein-protein interactions imex curation imex curation chattopadhyay-2006-6 in vitro In vitro fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret tag fluorescence tag visualisation by fluorescence tag fluorescence Chattopadhyay K., Bhatia S., Fiser A., Almo SC., Nathenson SG. Photobleaching-based FRET. nathenso@aecom.yu.edu,almo@aecom.yu.edu Journal of immunology (Baltimore, Md. : 1950) 2006 Only protein-protein interactions imex curation imex curation icosl_human ICOS ligand ICOSLG B7 homolog 2 B7-like protein Gl50 B7-related protein 1 B7H2 B7RP1 ICOSL KIAA0653 protein protein human Homo sapiens Human MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWSILAVLCLLVVVAVAIGWVCRDRCLQHSYAGAWAVSPETELTGHV icos_human Inducible T-cell costimulator ICOS AILIM Activation-inducible lymphocyte immunomediatory molecule protein protein human Homo sapiens Human MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL icoslg-5 3338352 N/A 3338358 unspecified role unspecified role neutral component neutral component purified purified signal+ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. Stoichiometry: 2.0 direct interaction direct interaction false false false figure 1D icoslg-7 3338352 N/A 3338358 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat c_terminus yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. Stoichiometry: 2.0 physical association physical association false false false figure 2C Cross-linking shows presence of ICOSL oligomers of higher stoichiometry. icoslg-8 3338352 N/A 3338358 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat c_terminus yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. Stoichiometry: 3.0 physical association physical association false false false figure 2Ci Cross-linking shows presence of ICOSL oligomers of higher stoichiometry. icoslg-9 3338352 N/A 3338358 unspecified role unspecified role neutral component neutral component subcellular prep subcellular preparation subcellular prep over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat c_terminus yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. Stoichiometry: 4.0 physical association physical association false false false figure 2Cii Cross-linking shows presence of ICOSL oligomers of higher stoichiometry. icos-icoslg-2 3338353 N/A 3338359 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat conditioned medium conditioned medium ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false B2M_signal tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. N/A 3338358 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat c_terminus yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false O75144:p.Tyr53Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Val62Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Tyr51Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. physical association physical association false false false figure 2A, 7A icos-icoslg-1 3338353 N/A 3338359 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat c_terminus yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. N/A 3338358 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat conditioned medium conditioned medium c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false IgV_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. physical association physical association false false false figure 3 B/C, 4A The IgV domain of ICOSL alone could bind to ICOS, however ICOSL-V-Ig was found to bind at least 8- to 10-fold less well to ICOS compared with ICOSL-VC-Ig. icos-icoslg-3 3338353 N/A 3338359 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat conditioned medium conditioned medium ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false B2M_signal tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. N/A 3338358 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat c_terminus yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false O75144:p.Tyr53Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Gln55Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Ser59Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Thr61Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Val62Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.[Gln55Ala;Val62Ala] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false O75144:p.Thr64Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.His66Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Val76Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Asp77Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Tyr80Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Ser87Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Leu47Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Tyr51Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Tyr53Phe mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Tyr51Phe mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Leu98Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Leu114Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Leu116Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false O75144:p.Leu120Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Gly121Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Phe122Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Gln123Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Glu124Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Phe138Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Pro141Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Val143Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Gly195Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Leu196Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Asn70Gln mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Asn102Gln mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false O75144:p.Asn137Gln mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Asn186Gln mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false O75144:p.Asn225Gln mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. physical association physical association false false false figure 6A, table 1 Disrupting mutations show equal or less than 5% binding. icoslg-4 3338354 N/A 3338359 unspecified role unspecified role bait bait conditioned medium conditioned medium ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false B2M_signal tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. N/A 3338358 unspecified role unspecified role prey prey purified purified signal+ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. physical association physical association false false false figure 1C monomeric ICOSL Both the oligomeric and monomeric forms of ICOSL-VC bound to soluble ICOS-Ig icos-icoslg-4 3338354 N/A 3338358 unspecified role unspecified role prey prey purified purified signal+ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. N/A 3338359 unspecified role unspecified role bait bait conditioned medium conditioned medium ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c_terminus fc-igg tag fc-igg tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false B2M_signal tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. N/A 3338358 unspecified role unspecified role prey prey purified purified signal+ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 3338444 3338438 physical association physical association false false false figure 1Ci oligomeric ICOSL Both the oligomeric and monomeric forms of ICOSL-VC bound to soluble ICOS-Ig. icoslg-icoslg-1 3338355 N/A 3338358 unspecified role unspecified role neutral component neutral component purified purified signal+ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. N/A 3338358 unspecified role unspecified role neutral component neutral component purified purified signal+ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. direct interaction direct interaction false false false figure 1B icoslg-1 3338356 N/A 3338358 unspecified role unspecified role neutral component neutral component purified purified signal+ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. N/A 3338358 unspecified role unspecified role neutral component neutral component purified purified signal+ecd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. direct interaction direct interaction false false false figure 1B (inset) icoslg-6 3338357 N/A 3338358 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat deltaC_construct sufficient to bind sufficient binding region sufficient to bind range range range range false c_terminus cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. N/A 3338358 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed n transfection treat nucleic acid transfection by treatment n transfection treat deltaC_construct sufficient to bind sufficient binding region sufficient to bind range range range range false c_terminus yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. physical association physical association false false false figure 2D-F,H.4 B/C The presence of ICOSL-YFP with a cysteine containing linker did not change the average FRET efficiency values of ICOSL. Fret efficiency was increased using an ICOSL construct missing the IgC domain, suggesting a higher degree of homo-association.