bhf-ucl saadi-2003-3 Dominant negative dimerization of a mutant homeodomain protein in Axenfeld-Rieger syndrome. in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa autoradiography autoradiography Saadi I., Kuburas A., Engle J.J., Russo A.F. Cardiac - Interactions involving cardiac related proteins GST tag in PITX2a is n-terminal and expressed in E. coli as described in ref. 4 (PMID: 9685346) PITX2a used in EMSA was also expressed in E. coli (ref. 4) andrew-russo@uiowa.edu Mol. Cell. Biol. (0270-7306) 2003 Only protein-protein interactions imex curation The authors have stated that PITX2a is of Human origin (Q99697-3) and that the origin of Pit1 is Rat. saadi-2003-1 Dominant negative dimerization of a mutant homeodomain protein in Axenfeld-Rieger syndrome. 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 Saadi I., Kuburas A., Engle J.J., Russo A.F. Cardiac - Interactions involving cardiac related proteins GST tag in PITX2a is n-terminal and expressed in E. coli as described in ref. 4 (PMID: 9685346) PITX2a used in EMSA was also expressed in E. coli (ref. 4) andrew-russo@uiowa.edu Mol. Cell. Biol. (0270-7306) 2003 Only protein-protein interactions imex curation The authors have stated that PITX2a is of Human origin (Q99697-3) and that the origin of Pit1 is Rat. saadi-2003-2 Dominant negative dimerization of a mutant homeodomain protein in Axenfeld-Rieger syndrome. in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Saadi I., Kuburas A., Engle J.J., Russo A.F. Cardiac - Interactions involving cardiac related proteins GST tag in PITX2a is n-terminal and expressed in E. coli as described in ref. 4 (PMID: 9685346) PITX2a used in EMSA was also expressed in E. coli (ref. 4) andrew-russo@uiowa.edu Mol. Cell. Biol. (0270-7306) 2003 Only protein-protein interactions imex curation The authors have stated that PITX2a is of Human origin (Q99697-3) and that the origin of Pit1 is Rat. q99697-3 Pituitary homeobox 2 ARP1A PITX2 ARP1 RGS RIEG RIEG1 ALL1-responsive protein ARP1 Homeobox protein PITX2 Paired-like homeodomain transcription factor 2 RIEG bicoid-related homeobox transcription factor Solurshin protein protein human Homo sapiens Human METNCRKLVSACVQLEKDKSQQGKNEDVGAEDPSKKKRQRRQRTHFTSQQLQELEATFQRNRYPDMSTREEIAVWTNLTEARVRVWFKNRRAKWRKRERNQQAELCKNGFGPQFNGLMQPYDDMYPGYSYNNWAAKGLTSASLSTKSFPFFNSMNVNPLSSQSMFSPPNSISSMSMSSSMVPSAVTGVPGSSLNSLNNLNNLSSPSLNSAVPTPACPYAPPTPPYVYRDTCNSSLASLRLKAKQHSSFGYASVQNPASNLSACQYAVDRPV Variant in position: 58:F->L (in ASGD4). bcd 2x pal 5n Bcd 2x pal 5n dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid synthetic construct synthetic construct GATCCATCTAATCCCGTCGGGATTAGATGGATC bcd 2x 5n Bcd 2x 5n dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid synthetic construct synthetic construct GATCCATCTAATCCCGTCGTAATCCGATGGATC bcd1x Bcd1x dna deoxyribonucleic acid DNA deoxyribonucleic acid dna DNA deoxyribonucleic acid synthetic construct synthetic construct GATCCGCACGGCCCATCTAATCCCGTGGGATC pit1_rat Pituitary-specific positive transcription factor 1 Pou1f1 Ghf-1 Pit-1 Pit1 Growth hormone factor 1 protein protein rat Rattus norvegicus (Rat) Rat MSCQPFTSADTFIPLNSDASAALPLRMHHSAAEGLPASNHATNVMSTATGLHYSVPSCHYGNQPSTYGVMAGTLTPCLYKFPDHTLSHGFPPLHQPLLAEDPTASEFKQELRRKSKLVEEPIDMDSPEIRELEQFANEFKVRRIKLGYTQTNVGEALAAVHGSEFSQTTICRFENLQLSFKNACKLKAILSKWLEEAEQVGALYNEKVGANERKRKRRTTISIAAKDALERHFGEHSKPSSQEIMRMAEELNLEKEVVRVWFCNRRQREKRVKTSLNQSLFSISKEHLECR bcd_2x_pal_5n-pitx2-1 1569531 n/a 1569534 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1569535 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct direct interaction direct interaction false false false Fig. 4B weak binding bcd_2x_5n-pitx2-6 1569531 n/a 1569534 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1569536 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct n/a 1569535 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct n/a 1569537 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct n/a 1569536 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct physical association physical association false false false Fig.5A, 5B bcd_2x_5n-pitx2-4 1569531 n/a 1569534 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1569536 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct n/a 1569536 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct n/a 1569537 competitor competitor competitor unspecified role unspecified role synthetic construct synthetic construct physical association physical association false false false Fig. 5A, 5C bcd1x-pitx2 1569531 n/a 1569534 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1569537 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 4B, 6A Lys 88 Glu increases dimer formation bcd1x-pitx2-1 1569531 n/a 1569534 unspecified role unspecified role prey prey Q99697-3:p.Lys88Arg mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 1569537 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct direct interaction direct interaction false false false Fig. 4B, 6A, 8B bcd_2x_5n-pitx2-7 1569531 n/a 1569534 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) n/a 1569536 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct direct interaction direct interaction false false false Fig.4B, 6A, 6B bcd_2x_5n-pitx2-8 1569531 n/a 1569534 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 n/a 1569536 unspecified role unspecified role bait bait region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false synthetic construct synthetic construct physical association physical association false false false Fig. 4B, 6A, 6B Lys 88 Glu mutant increases the dimer formation pitx2-pitx2-4 1569532 n/a 1569534 unspecified role unspecified role bait bait c-terminal hd sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false n/a 1569534 unspecified role unspecified role prey prey Q99697-3:p.Thr68Pro mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 1A, 1B, 2B pitx2-pitx2-5 1569532 n/a 1569534 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain false n/a 1569534 unspecified role unspecified role prey prey Q99697-3:p.Lys88Glu mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 2A The K88E mutant interaction with the the full length and the two C-terminal tail fragments showed a greater beta-galactosidase activity than the WT interaction. pitx2-pitx2-9 1569532 n/a 1569534 unspecified role unspecified role prey prey Q99697-3:p.Lys88Glu mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false Q99697-3:p.Lys88Arg mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false Q99697-3:p.Thr68Pro mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n/a 1569534 unspecified role unspecified role bait bait physical association physical association false false false Fig. 9A pitx2-pitx2-10 1569532 n/a 1569534 unspecified role unspecified role prey prey Q99697-3:p.Lys88Arg mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false Q99697-3:p.Thr68Pro mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q99697-3:p.Lys88Glu mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false n/a 1569534 unspecified role unspecified role bait bait Q99697-3:p.Lys88Arg mutation mutation certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 9A pitx2-pou1f1 1569532 n/a 1569538 unspecified role unspecified role prey prey n/a 1569534 unspecified role unspecified role bait bait Q99697-3:p.Lys88Glu mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q99697-3:p.Lys88Arg mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false Q99697-3:p.Thr68Pro mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 2C pitx2-pitx2-7 1569533 n/a 1569534 unspecified role unspecified role prey prey c-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c-terminus myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope 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 1569534 unspecified role unspecified role bait bait n-teminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false Q99697-3:p.Lys88Arg mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false Q99697-3:p.Lys88Glu mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) physical association physical association false false false Fig. 3A, 3C, 3D, 9B