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 S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Oncogene (0950-9232) 2007 imex curation Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu 12-Oct-2007: Contacted by JYOTI. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. orchard 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu Accepted 2007-OCT-13 by ORCHARD. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Oncogene (0950-9232) 2007 imex curation Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu 12-Oct-2007: Contacted by JYOTI. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. orchard Only protein-protein interactions chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu Accepted 2007-OCT-13 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Oncogene (0950-9232) 2007 imex curation Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu 12-Oct-2007: Contacted by JYOTI. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. orchard Only protein-protein interactions human-u2-os Human U2-OS osteosarcoma cell line u2-os U2-OS osteocaroma cell line Not in CAPRI and no primary literature reference anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu Accepted 2007-OCT-13 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Oncogene (0950-9232) 2007 imex curation Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu 12-Oct-2007: Contacted by JYOTI. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. orchard Only protein-protein interactions in vitro In vitro pull down pull down predetermined predetermined participant predetermined Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu Accepted 2007-OCT-13 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function. Oncogene (0950-9232) 2007 imex curation Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu 12-Oct-2007: Contacted by JYOTI. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. orchard 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 Chen D., Zhang Z., Li M., Wang W., Li Y., Rayburn ER., Hill DL., Wang H., Zhang R. Apoptosis - Interactions involving proteins with a function related to apoptosis An email from the authors confirmed that all recombinant proteins used were of human origin. hui.wang@ccc.uab.edu,ruiwen.zhang@ccc.uab.edu Accepted 2007-OCT-13 by ORCHARD. 12-Oct-2007: Contacted by JYOTI. A human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech) was used. Oncogene (0950-9232) 2007 orchard Only protein-protein interactions imex curation rl23_human 60S ribosomal protein L23 RPL23 60S ribosomal protein L17 Large ribosomal subunit protein uL14 protein protein human Homo sapiens Human MSKRGRGGSSGAKFRISLGLPVGAVINCADNTGAKNLYIISVKGIKGRLNRLPAAGVGDMVMATVKKGKPELRKKVHPAVVIRQRKSYRRKDGVFLYFEDNAGVIVNNKGEMKGSAITGPVAKECADLWPRIASNAGSIA DIP protein P62829 original sequence version: 116 807E14139B2EB0B5 mdm2_human E3 ubiquitin-protein ligase Mdm2 MDM2 p53-binding protein Mdm2 Oncoprotein Mdm2 Double minute 2 protein RING-type E3 ubiquitin transferase Mdm2 protein protein human Homo sapiens Human MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSENRCHLEGGSDQKDLVQELQEEKPSSSHLVSRPSTSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTGTPSNPDLDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP DIP protein Q00987 original sequence version: 93 DIP protein Q00987-1 original sequence version: 192 DIP protein Q00987 original sequence version: 88 DIP protein Q00987 original sequence version: 180 DIP protein Q00987 original sequence version: 188 DIP protein Q00987 original sequence version: 189 DIP protein Q00987 original sequence version: 115 DIP protein Q00987-1 original sequence version: null DIP protein Q00987 original sequence version: 149 DIP protein Q00987 original sequence version: 146 DIP protein Q00987 original sequence version: 140 DIP protein Q00987 original sequence version: 183 DIP protein Q00987 original sequence version: 145 DIP protein Q00987 original sequence version: 182 DIP protein Q00987 original sequence version: 151 DIP protein Q00987 original sequence version: 156 DIP protein Q00987-1 original sequence version: 160 DIP protein Q00987 original sequence version: 160 DIP protein Q00987 original sequence version: 168 DIP protein Q00987 original sequence version: 186 DIP protein Q00987 original sequence version: 206 F37CE163876BC983 rs7_human 40S ribosomal protein S7 RPS7 Small ribosomal subunit protein eS7 protein protein human Homo sapiens Human MFSSSAKIVKPNGEKPDEFESGISQALLELEMNSDLKAQLRELNITAAKEIEVGGGRKAIIIFVPVPQLKSFQKIQVRLVRELEKKFSGKHVVFIAQRRILPKPTRKSRTKNKQKRPRSRTLTAVHDAILEDLVFPSEIVGKRIRVKLDGSRLIKVHLDKAQQNNVEHKVETFSGVYKKLTGKDVNFEFPEFQL E21089929CC061E9 p53_human Cellular tumor antigen p53 TP53 P53 Tumor suppressor p53 Phosphoprotein p53 Antigen NY-CO-13 protein protein human Homo sapiens Human MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD DIP protein P04637 original sequence version: 121 DIP protein P04637 original sequence version: 115 DIP protein P04637-1 original sequence version: 224 DIP protein P04637 original sequence version: 117 DIP protein P04637-1 original sequence version: 226 DIP protein P04637 original sequence version: 226 DIP protein P04637 original sequence version: 212 DIP protein P04637 original sequence version: 220 DIP protein P04637-1 original sequence version: 220 DIP protein P04637-1 original sequence version: 145 DIP protein P04637 original sequence version: 146 DIP protein P04637 original sequence version: 145 DIP protein P04637 original sequence version: 166 DIP protein P04637-1 original sequence version: 166 DIP protein P04637 original sequence version: 169 DIP protein P04637 original sequence version: 177 DIP protein P04637 original sequence version: 175 DIP protein P04637-1 original sequence version: 175 DIP protein P04637-1 original sequence version: 172 DIP protein P04637-1 original sequence version: 169 DIP protein P04637 original sequence version: 170 DIP protein P04637 original sequence version: 172 DIP protein P04637-1 original sequence version: 194 DIP protein P04637 original sequence version: 196 DIP protein P04637 original sequence version: 185 DIP protein P04637-1 original sequence version: 191 DIP protein P04637 original sequence version: 191 DIP protein P04637 original sequence version: 180 DIP protein P04637-1 original sequence version: 197 DIP protein P04637 original sequence version: 183 DIP protein P04637-1 original sequence version: 183 DIP protein P04637-1 original sequence version: 185 DIP protein P04637 original sequence version: 199 DIP protein P04637 original sequence version: 201 DIP protein P04637 original sequence version: 189 DIP protein P04637 original sequence version: 235 DIP protein P04637 original sequence version: 230 DIP protein P04637 original sequence version: 234 DIP protein P04637-1 original sequence version: 234 DIP protein P04637-1 original sequence version: 235 DIP protein P04637 original sequence version: 239 DIP protein P04637-1 original sequence version: 212 DIP protein P04637-1 original sequence version: 213 DIP protein P04637 original sequence version: 213 DIP protein P04637 original sequence version: 206 DIP protein P04637-1 original sequence version: 188 DIP protein P04637-1 original sequence version: 228 DIP protein P04637 original sequence version: 208 DIP protein P04637 original sequence version: 240 DIP protein P04637-1 original sequence version: 240 DIP protein P04637 original sequence version: 204 DIP protein P04637-1 original sequence version: 204 DIP protein P04637-1 original sequence version: 209 DIP protein P04637-1 original sequence version: 203 DIP protein P04637 original sequence version: 221 DIP protein P04637-1 original sequence version: 152 DIP protein P04637 original sequence version: 217 DIP protein P04637-1 original sequence version: 140 DIP protein P04637 original sequence version: 215 DIP protein P04637 original sequence version: 214 DIP protein P04637-1 original sequence version: 182 DIP protein P04637 original sequence version: 182 DIP protein P04637 original sequence version: 188 DIP protein P04637 original sequence version: 192 DIP protein P04637-1 original sequence version: 192 DIP protein P04637 original sequence version: 218 DIP protein P04637-1 original sequence version: 218 AD5C149FD8106131 rl11_human 60S ribosomal protein L11 RPL11 CLL-associated antigen KW-12 Large ribosomal subunit protein uL5 protein protein human Homo sapiens Human MAQDQGEKENPMRELRIRKLCLNICVGESGDRLTRAAKVLEQLTGQTPVFSKARYTVRSFGIRRNEKIAVHCTVRGAKAEEILEKGLKVREYELRKNNFSDTGNFGFGIQEHIDLGIKYDPSIGIYGLDFYVVLGRPGFSIADKKRRTGCIGAKHRISKEEAMRWFQQKYDGIILPGK DIP protein P62913 original sequence version: null 26EC965C9239774E mdm2-rps7-2 1 8 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait physical association physical association we conducted a yeast two-hybrid assay using full-length MDM2 to screen a human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech, Mountain View, CA, USA). Sequence analysis of the candidate MDM2 interacting proteins identified RPL11 (Dai et al., 2006) and RPL23 (Dai et al., 2004), which have previously been shown to bind to MDM2. In addition, we identified 11 clones that encode a protein corresponding to full-length human S7. mdm2-rpl11-1 1 7 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association we conducted a yeast two-hybrid assay using full-length MDM2 to screen a human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech, Mountain View, CA, USA). Sequence analysis of the candidate MDM2 interacting proteins identified RPL11 (Dai et al., 2006) and RPL23 (Dai et al., 2004), which have previously been shown to bind to MDM2. In addition, we identified 11 clones that encode a protein corresponding to full-length human S7. mdm2-rpl23 1 7 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey physical association physical association we conducted a yeast two-hybrid assay using full-length MDM2 to screen a human prostate MATCHMAKER cDNA library (BD Biosciences, Clontech, Mountain View, CA, USA). Sequence analysis of the candidate MDM2 interacting proteins identified RPL11 (Dai et al., 2006) and RPL23 (Dai et al., 2004), which have previously been shown to bind to MDM2. In addition, we identified 11 clones that encode a protein corresponding to full-length human S7. mdm2-rps7-3 4 8 unspecified role unspecified role prey prey 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 direct interaction direct interaction In vitro pull-down assays demonstrated direct binding between MDM2 and S7; no co-factors were required. As shown in Figure 1b, GST-MDM2 (b1) or GST-S7 (b2), but not GST, bound to in vitro translated S7 or MDM2, respectively. 1 B1 rps7-mdm2 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 7 unspecified role unspecified role prey prey direct interaction direct interaction In vitro pull-down assays demonstrated direct binding between MDM2 and S7; no co-factors were required. As shown in Figure 1b, GST-MDM2 (b1) or GST-S7 (b2), but not GST, bound to in vitro translated S7 or MDM2, respectively. 1 B2 mdm2-rps7-4 5 8 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association full-length cDNAs of MDM2 and S7 were cloned into pGBKT7 and pGADT7 vectors, respectively, and co-transformed into the yeast host strain AH109. As expected, transformants had the capacity to grow on the yeast Minimal Synthetic Dropout (SD) plate, which lacked adenine (Ade), histidine (His), leucine (Leu) and tryptophan (Trp) (SD/-Ade/-His/-Leu/-Trp) 1 A, 1 E1, E2, E3 mdm2-rps7-6 2 8 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 region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 C2 when pCGT-T7-MDM2 and pCMV-Myc-S7 were co-transfected into COS7 cells, sequential immunoprecipitation (IP) using a Myc antibody (Figure 1c1) or T7 antibody (Figure 1c2) and immunoblotting assay with a T7 antibody or Myc antibody demonstrated binding between MDM2 and S7 Q00987; P62081 transfection rps7-mdm2-1 2 8 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 7 unspecified role unspecified role prey prey region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 C1 when pCGT-T7-MDM2 and pCMV-Myc-S7 were co-transfected into COS7 cells, sequential immunoprecipitation (IP) using a Myc antibody (Figure 1c1) or T7 antibody (Figure 1c2) and immunoblotting assay with a T7 antibody or Myc antibody demonstrated binding between MDM2 and S7 Q00987; P62081 transfection mdm2-rps7-1 3 8 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait physical association physical association 1 C2 Under normal physiological conditions, there was an interaction between endogenous MDM2 and S7, as indicated by immunoprecipitation using an MDM2 antibody (H221, Figure 1d1) or S7 antibody (Figure 1d2) followed by immunoblotting with an antibody against S7 (Figure 1d1) or MDM2 (SMP14, Figure 1d2). rps7-mdm2-2 3 8 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey physical association physical association 1 C1 Under normal physiological conditions, there was an interaction between endogenous MDM2 and S7, as indicated by immunoprecipitation using an MDM2 antibody (H221, Figure 1d1) or S7 antibody (Figure 1d2) followed by immunoblotting with an antibody against S7 (Figure 1d1) or MDM2 (SMP14, Figure 1d2). mdm2-rps7-5 5 8 unspecified role unspecified role prey prey region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 7 unspecified role unspecified role bait bait physical association physical association full-length cDNAs of MDM2 and S7 were cloned into pGBKT7 and pGADT7 vectors, respectively, and co-transformed into the yeast host strain AH109. As expected, transformants had the capacity to grow on the yeast Minimal Synthetic Dropout (SD) plate, which lacked adenine (Ade), histidine (His), leucine (Leu) and tryptophan (Trp) (SD/-Ade/-His/-Leu/-Trp) 1 A, 1 F1, F2, F3 mdm2-rps7-7 2 8 unspecified role unspecified role prey prey region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 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 region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association 1 G1 Q00987; P62081 transfection When full-length S7 and different mutants of MDM2 were co-transfected into COS7 cells, sequential immunoprecipitation and immunoblotting assays showed that the binding region in MDM2 is between aa 180-298 rps7-mdm2-3 2 8 unspecified role unspecified role bait bait region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 7 unspecified role unspecified role prey prey region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 G2 When full-length S7 and different mutants of MDM2 were co-transfected into COS7 cells, sequential immunoprecipitation and immunoblotting assays showed that the binding region in MDM2 is between aa 180�298 (Figure 1g1). Similarly, S7 aa 59�134 are responsible for the S7-MDM2 binding in COS7 cells (Figure 1g2). Q00987; P62081 transfection rps7-mdm2-4 2 8 unspecified role unspecified role bait bait region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 3 C1 Q00987; P62081 transfection Q00987; P62081 overexpressed rps7-mdm2-5 2 8 unspecified role unspecified role bait bait region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association 3 C1 Q00987; P62081; P04637 transfection HA-p53 was detected in GFPS7 immunoprecipitates of lysates from cells transfected with GFP-S7, T7-MDM2 and HA-p53, but not in cells overexpressed with one or two of these. This indicates the formation of a ternary complex among MDM2, S7 and p53. Q00987; P62081; P04637 overexpressed mdm2-rps7 3 8 unspecified role unspecified role prey prey 9 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait association association 3 D We also observed binding between MDM2 and p53 and between MDM2 and S7 when cells were exposed to low concentrations of actinomycin D (Act D) (5nM), which disrupts ribosomal biogenesis (Figure 3d). This likely results from the formation of a ternary complex among S7, MDM2 and p53 following the release of S7 in response to ribosomal stress. Actinomycin D (5 nM for 24 h) enhanced this interaction. This treatment causes ribosomal stress.