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/ Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Cell (0092-8674) 2007 imex curation Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov 01-April-2008: Contacted by JYOTI. Only protein-protein interactions 8 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. anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov Accepted 2008-MAR-6 by CDEROW. 01-April-2008: Contacted by JYOTI. Cell (0092-8674) 2007 Only protein-protein interactions 8 imex curation Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Cell (0092-8674) 2007 imex curation Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov 01-April-2008: Contacted by JYOTI. Only protein-protein interactions 8 human-jurkat Homo sapiens jurkat Human T cell leukemia ch-ip chromatin immunoprecipitation assay ch-ip primer specific pcr primer specific pcr Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis I don't think EMSA data can be captured as probes are not accurately described and are aimed at recognition sequences in multiple gene promoters Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov Accepted 2008-MAR-6 by CDEROW. 01-April-2008: Contacted by JYOTI. Cell (0092-8674) 2007 Only protein-protein interactions 8 imex curation Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Cell (0092-8674) 2007 imex curation Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov 01-April-2008: Contacted by JYOTI. Only protein-protein interactions 8 in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov Accepted 2008-MAR-6 by CDEROW. 01-April-2008: Contacted by JYOTI. Cell (0092-8674) 2007 Only protein-protein interactions 8 imex curation Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Cell (0092-8674) 2007 imex curation Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov 01-April-2008: Contacted by JYOTI. Only protein-protein interactions 8 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. tap tandem affinity purification TAP TAP tap ms participant Identification by mass spectrometry ms participant Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov Accepted 2008-MAR-6 by CDEROW. 01-April-2008: Contacted by JYOTI. Cell (0092-8674) 2007 Only protein-protein interactions 8 imex curation Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Cell (0092-8674) 2007 imex curation Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov 01-April-2008: Contacted by JYOTI. Only protein-protein interactions 8 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. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov Accepted 2008-MAR-6 by CDEROW. 01-April-2008: Contacted by JYOTI. Cell (0092-8674) 2007 Only protein-protein interactions 8 imex curation Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Cell (0092-8674) 2007 imex curation Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov 01-April-2008: Contacted by JYOTI. Only protein-protein interactions 8 human-jurkat Homo sapiens jurkat Human T cell leukemia anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov Accepted 2008-MAR-6 by CDEROW. 01-April-2008: Contacted by JYOTI. Cell (0092-8674) 2007 Only protein-protein interactions 8 imex curation Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Ribosomal Protein S3: A KH Domain Subunit in NF-kappaB Complexes that Mediates Selective Gene Regulation. Cell (0092-8674) 2007 imex curation Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov 01-April-2008: Contacted by JYOTI. Only protein-protein interactions 8 human-jurkat Homo sapiens jurkat Human T cell leukemia anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Wan F., Anderson D.E., Barnitz R.A., Snow A., Bidere N., Zheng L., Hegde V., Lam L.T., Staudt L.M., Levens D., Deutsch W.A., Lenardo M.J. Apoptosis - Interactions involving proteins with a function related to apoptosis Author states this in an indirect interaction no Cells were treated with 30 ng/ml TNFalpha, 30 min before lysis. Source of RelA inferred from previous work of donating author and confirmed by sequence length and mutant mapping. lenardo@nih.gov Accepted 2008-MAR-6 by CDEROW. 01-April-2008: Contacted by JYOTI. Cell (0092-8674) 2007 Only protein-protein interactions 8 imex curation rs3_human 40S ribosomal protein S3 RPS3 Small ribosomal subunit protein uS3 OK/SW-cl.26 protein protein human Homo sapiens Human MAVQISKKRKFVADGIFKAELNEFLTRELAEDGYSGVEVRVTPTRTEIIILATRTQNVLGEKGRRIRELTAVVQKRFGFPEGSVELYAEKVATRGLCAIAQAESLRYKLLGGLAVRRACYGVLRFIMESGAKGCEVVVSGKLRGQRAKSMKFVDGLMIHSGDPVNYYVDTAVRHVLLRQGVLGIKVKIMLPWDPTGKIGPKKPLPDHVSIVEPKDEILPTTPISEQKGGKPEPPAMPQPVPTA DIP protein P23396 original sequence version: 174 6ECBB34A8EE04AAF tf65_human Transcription factor p65 Nuclear factor NF-kappa-B p65 subunit RELA NFKB3 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 protein protein human Homo sapiens Human MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKRDLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTAELKICRVNRNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWEARGSFSQADVHRQVAIVFRTPPYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS DIP protein Q04206 original sequence version: 170 DIP protein Q04206 original sequence version: 173 DIP protein Q04206 original sequence version: 99 DIP protein Q04206-1 original sequence version: 99 DIP protein Q04206 original sequence version: 168 DIP protein Q04206 original sequence version: 128 DIP protein Q04206 original sequence version: 131 DIP protein Q04206 original sequence version: 153 DIP protein Q04206 original sequence version: 185 DIP protein Q04206 original sequence version: 181 DIP protein Q04206 original sequence version: 183 8147A6AF9F2445C6 ikba_human NF-kappa-B inhibitor alpha NFKBIA IKBA MAD3 NFKBI I-kappa-B-alpha Major histocompatibility complex enhancer-binding protein MAD3 protein protein human Homo sapiens Human MFQAAERPQEWAMEGPRDGLKKERLLDDRHDSGLDSMKDEEYEQMVKELQEIRLEPQEVPRGSEPWKQQLTEDGDSFLHLAIIHEEKALTMEVIRQVKGDLAFLNFQNNLQQTPLHLAVITNQPEIAEALLGAGCDPELRDFRGNTPLHLACEQGCLASVGVLTQSCTTPHLHSILKATNYNGHTCLHLASIHGYLGIVELLVSLGADVNAQEPCNGRTALHLAVDLQNPDLVSLLLKCGADVNRVTYQGYSPYQLTWGRPSTRIQQQLGQLTLENLQMLPESEDEESYDTESEFTEFTEDELPYDDCVFGGQRLTL DIP protein P25963 original sequence version: 160 DIP protein P25963 original sequence version: 157 DIP protein P25963 original sequence version: 145 DIP protein P25963 original sequence version: 170 DIP protein P25963 original sequence version: 122 DIP protein P25963 original sequence version: 128 DIP protein P25963 original sequence version: 78 DIP protein P25963 original sequence version: 121 DIP protein P25963 original sequence version: 124 088B313226786395 nfkb1_human Nuclear factor NF-kappa-B p105 subunit NFKB1 DNA-binding factor KBF1 EBP-1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 protein protein human Homo sapiens Human MAEDDPYLGRPEQMFHLDPSLTHTIFNPEVFQPQMALPTDGPYLQILEQPKQRGFRFRYVCEGPSHGGLPGASSEKNKKSYPQVKICNYVGPAKVIVQLVTNGKNIHLHAHSLVGKHCEDGICTVTAGPKDMVVGFANLGILHVTKKKVFETLEARMTEACIRGYNPGLLVHPDLAYLQAEGGGDRQLGDREKELIRQAALQQTKEMDLSVVRLMFTAFLPDSTGSFTRRLEPVVSDAIYDSKAPNASNLKIVRMDRTAGCVTGGEEIYLLCDKVQKDDIQIRFYEEEENGGVWEGFGDFSPTDVHRQFAIVFKTPKYKDINITKPASVFVQLRRKSDLETSEPKPFLYYPEIKDKEEVQRKRQKLMPNFSDSFGGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMKHGTMDTESKKDPEGCDKSDDKNTVNLFGKVIETTEQDQEPSEATVGNGEVTLTYATGTKEESAGVQDNLFLEKAMQLAKRHANALFDYAVTGDVKMLLAVQRHLTAVQDENGDSVLHLAIIHLHSQLVRDLLEVTSGLISDDIINMRNDLYQTPLHLAVITKQEDVVEDLLRAGADLSLLDRLGNSVLHLAAKEGHDKVLSILLKHKKAALLLDHPNGDGLNAIHLAMMSNSLPCLLLLVAAGADVNAQEQKSGRTALHLAVEHDNISLAGCLLLEGDAHVDSTTYDGTTPLHIAAGRGSTRLAALLKAAGADPLVENFEPLYDLDDSWENAGEDEGVVPGTTPLDMATSWQVFDILNGKPYEPEFTSDDLLAQGDMKQLAEDVKLQLYKLLEIPDPDKNWATLAQKLGLGILNNAFRLSPAPSKTLMDNYEVSGGTVRELVEALRQMGYTEAIEVIQAASSPVKTTSQAHSLPLSPASTRQQIDELRDSDSVCDSGVETSFRKLSFTESLTSGASLLTLNKMPHDYGQEGPLEGKI DIP protein P19838 original sequence version: 202 DIP protein P19838 original sequence version: 192 DIP protein P19838 original sequence version: 195 2A7C8FF86A10D1A8 il8_human_gene Human IL-8 gene gene gene human Homo sapiens Human il2ra_human_gene Human IL2RA (CD25) gene gene gene human Homo sapiens Human ikba_human_gene NF-kappa-B inhibitor alpha NFKBIA IKBA MAD3 NFKBI I-kappa-B-alpha Major histocompatibility complex enhancer-binding protein MAD3 gene gene human Homo sapiens Human cd69_human_gene Human CD69 gene gene gene human Homo sapiens Human il2_human_gene Human IL-2 gene IL2 gene gene human Homo sapiens Human rela-rps3-1 4 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait region tap tagged calmodulin binding peptide plus protein a tag CBP-ProtA tagged CBP-ProtA tagged tap tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association p65 Interacts with RPS3 Fig. 1A and Supp. Fig. 1 rela-rps3-2 3 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) 9 unspecified role unspecified role prey prey human-jurkat Homo sapiens jurkat Human T cell leukemia physical association physical association In a GST pull-down assay, we demonstrated that RPS3 binds to p65 Fig. 1B rela-rps3-3 6 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association Conversely, endogenous RPS3 was precipitated with antibodies against endogenous p65 and p50 (Figure 1D) Fig. 1D nfkb1-rps3-1 7 11 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association Lysates as in (C) were immunoblotted for RPS3 directly or after immunoprecipitation with p65 Fig. 1D The Santa Cruz catalogue suggests p50 Ab may only detect full length p105 NF-kB and not the p50 cleavage product so curated to parent protein only. rela-rps3-5 6 8 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey 9 unspecified role unspecified role prey prey 10 unspecified role unspecified role prey prey association association Immunoprecipitation of endogenous RPS3 revealed a strong association with endogenous p65 in Jurkat cells (Figure 1C). We also detected p50 and IκBα in this immunoprecipitate (Figure 1C). Fig. 1C The Santa Cruz catalogue suggests p50 Ab may only detect full length p105 NF-kB and not the p50 cleavage product so curated to parent protein only. rela-rps3-6 1 9 unspecified role unspecified role prey prey c-terminal region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range rela region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role bait bait physical association physical association We found that the N-terminal portion (NTD, aa 21�186) of the Rel homology domain (RHD) of p65 was necessary for binding to RPS3 Fig. 4E rela-rps3-7 5 8 unspecified role unspecified role bait bait n-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range kh region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role prey prey physical association physical association A similar analysis of RPS3 revealed that the N-terminal region (aa 1�111), particularly the KH domain (aa 41�111), was needed for p65 binding (Figure 4G). Fig. 4G rela-nfkbia-8 2 14 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait association association We found that precipitation of either p65 or RPS3 gave the same signal for the 200-300 bp region containing kappaB sites of two well-studied NF-kappaB target genes, IkappaBalpha and IL-8 (Figures 5A and S5). 50 ng/ml phorbol myristate acetate plus 1.5 uM ionomycin for 3 hr. Fig. 5A, 6A, 7B, 7C SiRNA studies show RPS3 and p65 bind in a mutually dependent fashion to kappaB sites in gene control sequences of IL-8 and NFKBIA antiCD3/IL2RA PCR shows binding was to the promoter region of the gene. rela-il8-1 2 9 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey association association We found that precipitation of either p65 or RPS3 gave the same signal for the 200-300 bp region containing kappaB sites of two well-studied NF-kappaB target genes, IkappaBalpha and IL-8 (Figures 5A and S5). 50 ng/ml phorbol myristate acetate plus 1.5 uM ionomycin for 3 hr. Fig. 5A, 6A, 7C SiRNA studies show RPS3 and p65 bind in a mutually dependent fashion to kappaB sites in gene control sequences of IL-8 and NFKBIA PCR shows binding was to the promoter region of the gene. rps3-il8-1 2 8 unspecified role unspecified role bait bait 12 unspecified role unspecified role prey prey association association We found that precipitation of either p65 or RPS3 gave the same signal for the 200-300 bp region containing kappaB sites of two well-studied NF-kappaB target genes, IkappaBalpha and IL-8 (Figures 5A and S5). 50 ng/ml phorbol myristate acetate plus 1.5 uM ionomycin for 3 hr. Fig. 5A, 6A SiRNA studies show RPS3 and p65 bind in a mutually dependent fashion to kappaB sites in gene control sequences of IL-8 and NFKBIA PCR shows binding was to the promoter region of the gene. rps3-nfkbia-3 2 14 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait association association We found that precipitation of either p65 or RPS3 gave the same signal for the 200-300 bp region containing kappaB sites of two well-studied NF-kappaB target genes, IkappaBalpha and IL-8 (Figures 5A and S5). 50 ng/ml phorbol myristate acetate plus 1.5 uM ionomycin for 3 hr. Fig. 5A, 6A SiRNA studies show RPS3 and p65 bind in a mutually dependent fashion to kappaB sites in gene control sequences of IL-8 and NFKBIA PCR shows binding was to the promoter region of the gene. rela-il2ra-1 2 9 unspecified role unspecified role bait bait 13 unspecified role unspecified role prey prey association association those in the CD25 and CD69 genes are occupied by p65 antiCD3/IL2RA Fig. 7b, 7C This gene does not bind RPS3 PCR shows binding was to the promoter region of the gene. rela-cd69-1 2 9 unspecified role unspecified role bait bait 15 unspecified role unspecified role prey prey association association those in the CD25 and CD69 genes are occupied by p65 antiCD3/IL2RA Fig. 7b, 7C This gene does not bind RPS3 PCR shows binding was to the promoter region of the gene. rela-il2-1 2 9 unspecified role unspecified role bait bait 16 unspecified role unspecified role prey prey association association We found that the p65 occupancy of kappaB sites in the promoters of IkappaBalpha, IL-8, and IL-2 was markedly reduced in RPS3-knockdown cells antiCD3/IL2RA Fig. 7b, 7C SiRNA studies suggest that occupancy of this gene promoter by TP65 requires RPS3. PCR shows binding was to the promoter region of the gene. rps3-il2-2 2 16 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait association association We found that the p65 occupancy of kappaB sites in the promoters of IkappaBalpha, IL-8, and IL-2 was markedly reduced in RPS3-knockdown cells antiCD3/IL2RA Fig. 7b, 7C SiRNA studies suggest that occupancy of this gene promoter by TP65 requires RPS3. PCR shows binding was to the promoter region of the gene. rps3-nfkbia-4 2 14 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 50 ng/ml phorbol myristate acetate plus 1.5 uM ionomycin for 3 hr. Supp. Fig. 5 PCR suggests RPS3 is recruited to the kappaB site of the NFKBIA gene PCR shows binding was to the promoter region of the gene. rps3-il8-2 2 12 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 50 ng/ml phorbol myristate acetate plus 1.5 uM ionomycin for 3 hr. Supp. Fig. 5 PCR suggests RPS3 binds to the kappaB region of the IL-8 gene PCR shows binding was to the promoter region of the gene. rela-rps3-4 6 8 unspecified role unspecified role prey prey 11 unspecified role unspecified role bait bait physical association physical association Conversely, endogenous RPS3 was precipitated with antibodies against endogenous p65 and p50 (Figure 1D) Fig. 1D The Santa Cruz catalogue suggests p50 Ab may only detect full length p105 NF-kB and not the p50 cleavage product so curated to parent protein only.