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/ Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Nature (0028-0836) 2001 Gray WM., Kepinski S., Rouse D., Leyser O., Estelle M. The authors state that "the ASK2 antibody cross-reacts with the co-migrating GST protein" and coclude that "we could not confirm that ASK1 and ASK2 were missing from the GST control". Because of this the ASK1 and ASK2 proteins were not curated. Note the parent term for Participant Detection 'western blot' is used to allow interaction detected by anti-myc (TIR1) and anti-CUL1 (CUL1) detection to be curated in this entry Transgenic Arabidopsis plants expressing an myc tagged TIR1 protein construct were used in this study. arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Gray WM., Kepinski S., Rouse D., Leyser O., Estelle M. The authors state that "the ASK2 antibody cross-reacts with the co-migrating GST protein" and coclude that "we could not confirm that ASK1 and ASK2 were missing from the GST control". Because of this the ASK1 and ASK2 proteins were not curated. Note the parent term for Participant Detection 'western blot' is used to allow interaction detected by anti-myc (TIR1) and anti-CUL1 (CUL1) detection to be curated in this entry Transgenic Arabidopsis plants expressing an myc tagged TIR1 protein construct were used in this study. Accepted 2008-JUN-13 by JYOTI. Nature (0028-0836) 2001 Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Nature (0028-0836) 2001 Gray WM., Kepinski S., Rouse D., Leyser O., Estelle M. The authors state that "the ASK2 antibody cross-reacts with the co-migrating GST protein" and coclude that "we could not confirm that ASK1 and ASK2 were missing from the GST control". Because of this the ASK1 and ASK2 proteins were not curated. Note the parent term for Participant Detection 'western blot' is used to allow interaction detected by anti-myc (TIR1) and anti-CUL1 (CUL1) detection to be curated in this entry Transgenic Arabidopsis plants expressing an myc tagged TIR1 protein construct were used in this study. in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Gray WM., Kepinski S., Rouse D., Leyser O., Estelle M. The authors state that "the ASK2 antibody cross-reacts with the co-migrating GST protein" and coclude that "we could not confirm that ASK1 and ASK2 were missing from the GST control". Because of this the ASK1 and ASK2 proteins were not curated. Note the parent term for Participant Detection 'western blot' is used to allow interaction detected by anti-myc (TIR1) and anti-CUL1 (CUL1) detection to be curated in this entry Transgenic Arabidopsis plants expressing an myc tagged TIR1 protein construct were used in this study. Accepted 2008-JUN-13 by JYOTI. Nature (0028-0836) 2001 Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Nature (0028-0836) 2001 Gray WM., Kepinski S., Rouse D., Leyser O., Estelle M. The authors state that "the ASK2 antibody cross-reacts with the co-migrating GST protein" and coclude that "we could not confirm that ASK1 and ASK2 were missing from the GST control". Because of this the ASK1 and ASK2 proteins were not curated. Note the parent term for Participant Detection 'western blot' is used to allow interaction detected by anti-myc (TIR1) and anti-CUL1 (CUL1) detection to be curated in this entry Transgenic Arabidopsis plants expressing an myc tagged TIR1 protein construct were used in this study. in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Gray WM., Kepinski S., Rouse D., Leyser O., Estelle M. The authors state that "the ASK2 antibody cross-reacts with the co-migrating GST protein" and coclude that "we could not confirm that ASK1 and ASK2 were missing from the GST control". Because of this the ASK1 and ASK2 proteins were not curated. Note the parent term for Participant Detection 'western blot' is used to allow interaction detected by anti-myc (TIR1) and anti-CUL1 (CUL1) detection to be curated in this entry Transgenic Arabidopsis plants expressing an myc tagged TIR1 protein construct were used in this study. Accepted 2008-JUN-13 by JYOTI. Nature (0028-0836) 2001 Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Nature (0028-0836) 2001 Gray WM., Kepinski S., Rouse D., Leyser O., Estelle M. The authors state that "the ASK2 antibody cross-reacts with the co-migrating GST protein" and coclude that "we could not confirm that ASK1 and ASK2 were missing from the GST control". Because of this the ASK1 and ASK2 proteins were not curated. Note the parent term for Participant Detection 'western blot' is used to allow interaction detected by anti-myc (TIR1) and anti-CUL1 (CUL1) detection to be curated in this entry Transgenic Arabidopsis plants expressing an myc tagged TIR1 protein construct were used in this study. in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Gray WM., Kepinski S., Rouse D., Leyser O., Estelle M. The authors state that "the ASK2 antibody cross-reacts with the co-migrating GST protein" and coclude that "we could not confirm that ASK1 and ASK2 were missing from the GST control". Because of this the ASK1 and ASK2 proteins were not curated. Note the parent term for Participant Detection 'western blot' is used to allow interaction detected by anti-myc (TIR1) and anti-CUL1 (CUL1) detection to be curated in this entry Transgenic Arabidopsis plants expressing an myc tagged TIR1 protein construct were used in this study. Accepted 2008-JUN-13 by JYOTI. Nature (0028-0836) 2001 cul1_arath Cullin-1 CUL1 At4g02570 T10P11.26 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MERKTIDLEQGWDYMQTGITKLKRILEGLNEPAFDSEQYMMLYTTIYNMCTQKPPHDYSQQLYDKYREAFEEYINSTVLPALREKHDEFMLRELFKRWSNHKVMVRWLSRFFYYLDRYFIARRSLPPLNEVGLTCFRDLVYNELHSKVKQAVIALVDKEREGEQIDRALLKNVLDIYVEIGMGQMERYEEDFESFMLQDTSSYYSRKASSWIQEDSCPDYMLKSEECLKKERERVAHYLHSSSEPKLVEKVQHELLVVFASQLLEKEHSGCRALLRDDKVDDLSRMYRLYHKILRGLEPVANIFKQHVTAEGNALVQQAEDTATNQVANTASVQEQVLIRKVIELHDKYMVYVTECFQNHTLFHKALKEAFEIFCNKTVAGSSSAELLATFCDNILKKGGSEKLSDEAIEDTLEKVVKLLAYISDKDLFAEFYRKKLARRLLFDRSANDDHERSILTKLKQQCGGQFTSKMEGMVTDLTLARENQNSFEDYLGSNPAANPGIDLTVTVLTTGFWPSYKSFDINLPSEMIKCVEVFKGFYETKTKHRKLTWIYSLGTCHINGKFDQKAIELIVSTYQAAVLLLFNTTDKLSYTEILAQLNLSHEDLVRLLHSLSCAKYKILLKEPNTKTVSQNDAFEFNSKFTDRMRRIKIPLPPVDERKKVVEDVDKDRRYAIDAAIVRIMKSRKVLGHQQLVSECVEQLSRMFKPDIKAIKKRMEDLITRDYLERDKENPNMFRYLA DIP protein Q94AH6 original sequence version: 102 DIP protein Q94AH6 original sequence version: 95 45041357DE8DAD8C tir1_arath Protein TRANSPORT INHIBITOR RESPONSE 1 TIR1 FBL1 WEI1 T20O10.80 At3g62980 Weak ethylene-insensitive protein 1 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MQKRIALSFPEEVLEHVFSFIQLDKDRNSVSLVCKSWYEIERWCRRKVFIGNCYAVSPATVIRRFPKVRSVELKGKPHFADFNLVPDGWGGYVYPWIEAMSSSYTWLEEIRLKRMVVTDDCLELIAKSFKNFKVLVLSSCEGFSTDGLAAIAATCRNLKELDLRESDVDDVSGHWLSHFPDTYTSLVSLNISCLASEVSFSALERLVTRCPNLKSLKLNRAVPLEKLATLLQRAPQLEELGTGGYTAEVRPDVYSGLSVALSGCKELRCLSGFWDAVPAYLPAVYSVCSRLTTLNLSYATVQSYDLVKLLCQCPKLQRLWVLDYIEDAGLEVLASTCKDLRELRVFPSEPFVMEPNVALTEQGLVSVSMGCPKLESVLYFCRQMTNAALITIARNRPNMTRFRLCIIEPKAPDYLTLEPLDIGFGAIVEHCKDLRRLSLSGLLTDKVFEYIGTYAKKMEMLSVAFAGDSDLGMHHVLSGCDSLRKLEIRDCPFGDKALLANASKLETMRSLWMSSCSVSFGACKLLGQKMPKLNVEVIDERGAPDSRPESCPVERVFIYRTVAGPRFDMPGFVWNMDQDSTMRFSRQIITTNGL DIP protein Q570C0 original sequence version: 83 DIP protein Q570C0 original sequence version: 40 DIP protein Q570C0 original sequence version: 86 DIP protein Q570C0 original sequence version: 60 9E19ED5DABF40D07 iaa7_arath Auxin-responsive protein IAA7 Indoleacetic acid-induced protein 7 Auxin resistant 2 IAA7 AXR2 At3g23050 MXC7.8 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MIGQLMNLKATELCLGLPGGAEAVESPAKSAVGSKRGFSETVDLMLNLQSNKEGSVDLKNVSAVPKEKTTLKDPSKPPAKAQVVGWPPVRNYRKNMMTQQKTSSGAEEASSEKAGNFGGGAAGAGLVKVSMDGAPYLRKVDLKMYKSYQDLSDALAKMFSSFTMGNYGAQGMIDFMNESKLMNLLNSSEYVPSYEDKDGDWMLVGDVPWEMFVESCKRLRIMKGSEAVGLAPRAMEKYCKNRS DIP protein Q38825 original sequence version: 111 DIP protein Q38825 original sequence version: 70 DIP protein Q38825 original sequence version: 88 830E9B9EA2C2B5D0 iaa17_arath Auxin-responsive protein IAA17 Indoleacetic acid-induced protein 17 Auxin response 3 IAA17 AXR3 At1g04250 F19P19.31 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MMGSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLNNEPANKEGSTTHDVVTFDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVKVSMDGAPYLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFMNERKLMDLVNSWDYVPSYEDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAMEKCKSRA DIP protein P93830 original sequence version: null ECEA39207476581E iaa7-tir1 1 6 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role bait bait physical association physical association Figure 4a To examine whether SCFTIR1 physically interacts with AUX/ IAA proteins, the AXR2 antibody was used in immunoprecipitation experiments with extracts prepared from seedlings expressing the c-myc epitope-tagged TIR1 derivative. TIR1 Myc was readily detected in anti-AXR2 immunoprecipitates but was absent from control precipitations using the AXR2 pre-immune serum iaa7-tir1-cul1 3 6 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress 7 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 Q38825:p.Pro87Ser mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain P S Wed May 02 19:32:48 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'pro87ser' Tue Jun 30 16:25:44 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Pro87Ser' ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role prey prey association association Figure 4b TIR1�Myc and AtCUL1 both co-puri�ed with the GST�AXR2 fusion protein but were absent in control pulldown assays using GST alone Details of the mutation in the mutatnt axr2-1 were taken from sequence provided by Nagpal et al (PMID 10859186) which was provided as a reference in the text. The coordinates were derived from the UniProt record for thie protein. iaa17-tir1-1 2 6 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress 8 unspecified role unspecified role bait bait P93830:p.Pro88Leu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain P L Wed May 02 15:27:45 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'pro88leu' Tue Jun 30 17:53:43 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Pro88Leu' 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 Figure 4c To determine whether SCFTIR1 interacts with additional AUX/ IAA proteins, we tested GST�AXR3 in pull-down assays. Similar to the results obtained with AXR2, GST�AXR3 co-puri�ed with TIR1 protein and the axr3-1 mutation substantially disrupted this interaction Details of the mutation in the mutatnt axr3-1 were taken from sequence provided by Nagpal et al (PMID 10859186) which was provided as a reference in the text. The coordinates were derived from the UniProt record for thie protein. iaa7-tir1-1 2 6 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress 7 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association Figure 4d TIR1 was able to interact, albeit at a reduced level, with a GST�AXR2 fusion protein containing only domain II (AXR271�100). In contrast, when a short deletion was introduced into the highly conserved core of domain II, this mutant derivative of AXR2 (AXR2D86�88) interacted very weakly with TIR1 (Fig. 4d). These data demonstrate that domain II is both necessary and suf�cient to bind SCFTIR1. iaa7-tir1-2 4 7 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) 6 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress physical association physical association Figure 5 Applied auxin also promoted the SCFTIR1�AUX/IAA interaction in a dose-dependent manner (Fig. 5b). Auxin treatment enhanced the SCFTIR1�AXR2/AXR3 interaction at concentrations as low as 0.5 mM. The authors noted that auxin treatment enhanced the TIR1 AXR2 interaction at concentrations as low as 0.5 micro molar. 2,4-dichlorophenoxy acetic acid (2,4-D) treatments. iaa17-tir1-2 4 8 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) 6 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress physical association physical association Figure 5 Auxin treatment enhanced the SCFTIR1�AXR2/AXR3 interaction at concentrations as low as 0.5micro molar. The authors noted that auxin treatment enhanced the TIR1 AXR2 interaction at concentrations as low as 0.5 micro molar. 2,4-dichlorophenoxy acetic acid (2,4-D) treatment.