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/ Synergistic activation of seed storage protein gene expression in Arabidopsis by ABI3 and two bZIPs related to OPAQUE2. Synergistic activation of seed storage protein gene expression in Arabidopsis by ABI3 and two bZIPs related to OPAQUE2. J. Biol. Chem. (0021-9258) 2003 imex curation Lara P., Onate-Sanchez L., Abraham Z., Ferrandiz C., Diaz I., Carbonero P., Vicente-Carbajosa J. Assays were carried out at 23C and 30C. jvicente@bit.etsia.upm.es. 14-May-2008: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa predetermined predetermined participant predetermined Lara P., Onate-Sanchez L., Abraham Z., Ferrandiz C., Diaz I., Carbonero P., Vicente-Carbajosa J. Assays were carried out at 23C and 30C. jvicente@bit.etsia.upm.es. Accepted 2008-MAY-2 by CDEROW. 14-May-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2003 Only protein-protein interactions imex curation Synergistic activation of seed storage protein gene expression in Arabidopsis by ABI3 and two bZIPs related to OPAQUE2. Synergistic activation of seed storage protein gene expression in Arabidopsis by ABI3 and two bZIPs related to OPAQUE2. J. Biol. Chem. (0021-9258) 2003 imex curation Lara P., Onate-Sanchez L., Abraham Z., Ferrandiz C., Diaz I., Carbonero P., Vicente-Carbajosa J. Assays were carried out at 23C and 30C. jvicente@bit.etsia.upm.es. 14-May-2008: 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 Lara P., Onate-Sanchez L., Abraham Z., Ferrandiz C., Diaz I., Carbonero P., Vicente-Carbajosa J. Assays were carried out at 23C and 30C. jvicente@bit.etsia.upm.es. Accepted 2008-MAY-2 by CDEROW. 14-May-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2003 Only protein-protein interactions imex curation cru3-arath-dna CRU3 promoter dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress TAATCCTACGTGGCTCA 4F7724F865B735FD op2_maize Regulatory protein opaque-2 O2 protein protein maize Zea mays (Maize) Maize MEHVISMEEILGPFWELLPPPAPEPEREQPPVTGIVVGSVIDVAAAGHGDGDMMDQQHATEWTFERLLEEEALTTSTPPPVVVVPNSCCSGALNADRPPVMEEAVTMAPAAVSSAVVGDPMEYNAILRRKLEEDLEAFKMWRADSSVVTSDQRSQGSNNHTGGSSIRNNPVQNKLMNGEDPINNNHAQTAGLGVRLATSSSSRDPSPSDEDMDGEVEILGFKMPTEERVRKKESNRESARRSRYRKAAHLKELEDQVAQLKAENSCLLRRIAALNQKYNDANVDNRVLRADMETLRAKVKMGEDSLKRVIEMSSSVPSSMPISAPTPSSDAPVPPPPIRDSIVGYFSATAADDDASVGNGFLRLQAHQEPASMVVGGTLSATEMNRVAAATHCAGAMELIQTAMGSMPPTSASGSTPPPQIMSCWVQMGPYTWTCIRHCGFRDRWEHFICRRR 513A8AB8D5ABD999 bzp10_arath Basic leucine zipper 10 BZIP10 BZO2H1 Basic leucine zipper OPAQUE 2 homolog 1 T10P11.9 At4g02640 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MNSIFSIDDFSDPFWETPPIPLNPDSSKPVTADEVSQSQPEWTFEMFLEEISSSAVSSEPLGNNNNAIVGVSSAQSLPSVSGQNDFEDDSRFRDRDSGNLDCAAPMTTKTVIVDSDDYRRVLKNKLETECATVVSLRVGSVKPEDSTSSPETQLQPVQSSPLTQGELGVTSSLPAEVKKTGVSMKQVTSGSSREYSDDEDLDEENETTGSLKPEDVKKSRRMLSNRESARRSRRRKQEQTSDLETQVNDLKGEHSSLLKQLSNMNHKYDEAAVGNRILKADIETLRAKVKMAEETVKRVTGMNPMLLGRSSGHNNNNRMPITGNNRMDSSSIIPAYQPHSNLNHMSNQNIGIPTILPPRLGNNFAAPPSQTSSPLQRIRNGQNHHVTPSANPYGWNTEPQNDSAWPKKCVD 9C651436D8D167CF abi3_arath B3 domain-containing transcription factor ABI3 ABI3 MSD24.2 At3g24650 Protein ABSCISIC ACID-INSENSITIVE 3 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MKSLHVAANAGDLAEDCGILGGDADDTVLMDGIDEVGREIWLDDHGGDNNHVHGHQDDDLIVHHDPSIFYGDLPTLPDFPCMSSSSSSSTSPAPVNAIVSSASSSSAASSSTSSAASWAILRSDGEDPTPNQNQYASGNCDDSSGALQSTASMEIPLDSSQGFGCGEGGGDCIDMMETFGYMDLLDSNEFFDTSAIFSQDDDTQNPNLMDQTLERQEDQVVVPMMENNSGGDMQMMNSSLEQDDDLAAVFLEWLKNNKETVSAEDLRKVKIKKATIESAARRLGGGKEAMKQLLKLILEWVQTNHLQRRRTTTTTTNLSYQQSFQQDPFQNPNPNNNNLIPPSDQTCFSPSTWVPPPPQQQAFVSDPGFGYMPAPNYPPQPEFLPLLESPPSWPPPPQSGPMPHQQFPMPPTSQYNQFGDPTGFNGYNMNPYQYPYVPAGQMRDQRLLRLCSSATKEARKKRMARQRRFLSHHHRHNNNNNNNNNNQQNQTQIGETCAAVAPQLNPVATTATGGTWMYWPNVPAVPPQLPPVMETQLPTMDRAGSASAMPRQQVVPDRRQGWKPEKNLRFLLQKVLKQSDVGNLGRIVLPKKEAETHLPELEARDGISLAMEDIGTSRVWNMRYRFWPNNKSRMYLLENTGDFVKTNGLQEGDFIVIYSDVKCGKYLIRGVKVRQPSGQKPEAPPSSAATKRQNKSQRNINNNSPSANVVVASPTSQTVK D6A0F740D733060F at2s1-arath-dna At2S1 promoter dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress TTCTTACACGTGAACTC 4DB83962989C3767 bzp25_arath Basic leucine zipper 25 BZIP25 BZO2H4 Basic leucine zipper OPAQUE 2 homolog 4 T14E10.190 At3g54620 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MHIVFSVDDLTESFWPVPAPAPSPGSSSTPSPTQNVADGMTRSQSEWAFHRLINELSGSDSSPTTNTIERSPPPVQSLSRLEETVDETEDVVEIQKPQNHRRLPVDDQGKNRNRAPSSDPVDSSAPVVVDPNQYHAILKSKLELACAAVARRVGTVKPEDSSASASNQKQAQGSIVAQTSPGASSVRFSPTTSTQKKPDVPARQTSISSRDDSDDDDLDGDADNGDPTDVKRARRMLSNRESARRSRRRKQEQMNEFDTQVGQLRAEHSTLINRLSDMNHKYDAAAVDNRILRADIETLRTKVKMAEETVKRVTGVNPLHWSRPNMGIPFSNTPSASSSIPPNSNHILKPANSSTNTSAGLAQNQRVETANFLPEQVNREGMQNPFAPDSNLYETLPHWNHKH 1AF17B764F03F5A1 at4g02640-abi3 2 6 unspecified role unspecified role prey prey n terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role bait bait n terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 5B The results of such analyses are displayed in Fig. 5B, showing that AtbZIP10 and ABI3 are able to interact through the Nterminal regions of both proteins. at3g54620-abi3-1 2 6 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 5B Likewise, an interaction between the ABI3 N and AtbZIP25 C-terminal region was also detected. Interactions between AtbZIP25 and ABI3 could not be reliably reproduced at 30 C, but lowering incubation temperatures to 23 C resulted in consistent data in all replicated experiments. From Figure 5B it would seem that both N and C terminal regions of AtZIP25 interact with ABI3. at3g54620-dna 1 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 4B In Fig. 4B the results of EMSA demonstrate that the AtbZIP10 and AtbZIP25 binding to these sequences occurs specifically through the ACGT boxes, since mutations (AaGg) introduced at this core abolished the interaction in the context of both At21S and CRU3 promoters. Mutation of the ACGT sequence to AAGG abolished interaction. The protein was also synthesised in-vitro but it is not possible to tell if this was done, as well as ecoli expression, in this case. at3g54620-dna-1 1 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 3 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 4B In Fig. 4B the results of EMSA demonstrate that the AtbZIP10 and AtbZIP25 binding to these sequences occurs specifically through the ACGT boxes, since mutations (AaGg) introduced at this core abolished the interaction in the context of both At21S and CRU3 promoters. Mutation of the ACGT sequence to AAGG abolished interaction. The protein was also synthesised in-vitro but it is not possible to tell if this was done, as well as ecoli expression, in this case. at4g02640-dna-2 1 5 unspecified role unspecified role neutral component neutral component ecobb Escherichia coli (strain B / BL21) 3 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 4B In Fig. 4B the results of EMSA demonstrate that the AtbZIP10 and AtbZIP25 binding to these sequences occurs specifically through the ACGT boxes, since mutations (AaGg) introduced at this core abolished the interaction in the context of both At21S and CRU3 promoters. Mutation of the ACGT sequence to AAGG abolished interaction. at4g02640-dna-3 1 5 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 4B and Figure 4C In Fig. 4B the results of EMSA demonstrate that the AtbZIP10 and AtbZIP25 binding to these sequences occurs specifically through the ACGT boxes, since mutations (AaGg) introduced at this core abolished the interaction in the context of both At21S and CRU3 promoters. Mutation of the ACGT sequence to AAGG abolished interaction. The protein was also synthesised in-vitro but it is not possible to tell if this was done, as well as ecoli expression, in this case. The presence of the ABI3 (Q01593) protein stimulated protein binding to DNA It is not clear from the text if recombinant or in vitro synthesised proteins were used o2-dna-4 1 7 unspecified role unspecified role neutral component neutral component 4 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 4C EMSA experiments shown in Fig. 4C, display the binding behavior of different amounts of AtbZIP10 or O2 proteins incubated with target DNA sequences. A clear increase in the binding of both proteins to their target sites was obtained in the presence of ABI3 The presence of the ABI3 (Q01593) protein increased protein binding to DNA It is not clear from the text if recombinant or in vitro synthesised proteins were used.