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/ http://www.ebi.ac.uk/ trazzi-2009-1 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 anti tag western anti tag western blot anti tag western Trazzi S., Perini G., Bernardoni R., Zoli M., Reese JC., Musacchio A., Della Valle G. CHIP reported in Figure 3 were not annotated since they reported the interactions with two aspecific DNA sequences. Interaction with Histone 3 were not captured since curator cannot assign a unique identifier to this interactor. giuliano.dellavalle@unibo.it PloS one 2009 imex curation Only protein-protein interactions imex curation trazzi-2009-2 in vitro In vitro crosslink cross-linking study crosslink anti tag western anti tag western blot anti tag western Trazzi S., Perini G., Bernardoni R., Zoli M., Reese JC., Musacchio A., Della Valle G. CHIP reported in Figure 3 were not annotated since they reported the interactions with two aspecific DNA sequences. Interaction with Histone 3 were not captured since curator cannot assign a unique identifier to this interactor. giuliano.dellavalle@unibo.it PloS one 2009 imex curation Only protein-protein interactions imex curation trazzi-2009-3 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 Trazzi S., Perini G., Bernardoni R., Zoli M., Reese JC., Musacchio A., Della Valle G. CHIP reported in Figure 3 were not annotated since they reported the interactions with two aspecific DNA sequences. Interaction with Histone 3 were not captured since curator cannot assign a unique identifier to this interactor. giuliano.dellavalle@unibo.it PloS one 2009 imex curation Only protein-protein interactions imex curation trazzi-2009-4 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. bifc bimolecular fluorescence complementation bifc tag fluorescence tag visualisation by fluorescence tag fluorescence Trazzi S., Perini G., Bernardoni R., Zoli M., Reese JC., Musacchio A., Della Valle G. CHIP reported in Figure 3 were not annotated since they reported the interactions with two aspecific DNA sequences. Interaction with Histone 3 were not captured since curator cannot assign a unique identifier to this interactor. giuliano.dellavalle@unibo.it PloS one 2009 imex curation Only protein-protein interactions imex curation cenpc_human Centromere protein C CENPC CENPC1 Centromere autoantigen C Centromere protein C 1 ICEN7 Interphase centromere complex protein 7 protein protein human Homo sapiens Human MAASGLDHLKNGYRRRFCRPSRARDINTEQGQNVLEILQDCFEEKSLANDFSTNSTKSVPNSTRKIKDTCIQSPSKECQKSHPKSVPVSSKKKEASLQFVVEPSEATNRSVQAHEVHQKILATDVSSKNTPDSKKISSRNINDHHSEADEEFYLSVGSPSVLLDAKTSVSQNVIPSSAQKRETYTFENSVNMLPSSTEVSVKTKKRLNFDDKVMLKKIEIDNKVSDEEDKTSEGQERKPSGSSQNRIRDSEYEIQRQAKKSFSTLFLETVKRKSESSPIVRHAATAPPHSCPPDDTKLIEDEFIIDESDQSFASRSWITIPRKAGSLKQRTISPAESTALLQGRKSREKHHNILPKTLANDKHSHKPHPVETSQPSDKTVLDTSYALIGETVNNYRSTKYEMYSKNAEKPSRSKRTIKQKQRRKFMAKPAEEQLDVGQSKDENIHTSHITQDEFQRNSDRNMEEHEEMGNDCVSKKQMPPVGSKKSSTRKDKEESKKKRFSSESKNKLVPEEVTSTVTKSRRISRRPSDWWVVKSEESPVYSNSSVRNELPMHHNSSRKSTKKTNQSSKNIRKKTIPLKRQKTATKGNQRVQKFLNAEGSGGIVGHDEISRCSLSEPLESDEADLAKKKNLDCSRSTRSSKNEDNIMTAQNVPLKPQTSGYTCNIPTESNLDSGEHKTSVLEESGPSRLNNNYLMSGKNDVDDEEVHGSSDDSKQSKVIPKNRIHHKLVLPSNTPNVRRTKRTRLKPLEYWRGERIDYQGRPSGGFVISGVLSPDTISSKRKAKENIGKVNKKSNKKRICLDNDERKTNLMVNLGIPLGDPLQPTRVKDPETREIILMDLVRPQDTYQFFVKHGELKVYKTLDTPFFSTGKLILGPQEEKGKQHVGQDILVFYVNFGDLLCTLHETPYILSTGDSFYVPSGNYYNIKNLRNEESVLLFTQIKR cenpa_human Histone H3-like centromeric protein A CENPA Centromere autoantigen A Centromere protein A protein protein human Homo sapiens Human MGPRRRSRKPEAPRRRSPSPTPTPGPSRRGPSLGASSHQHSRRRQGWLKEIRKLQKSTHLLIRKLPFSRLAREICVKFTRGVDFNWQAQALLALQEAAEAFLVHLFEDAYLLTLHAGRVTLFPKDVQLARRIRGLEEGLG canpc-1 3493630 N/A 3493634 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false N/A 3493634 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false flag tag 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 false physical association physical association false false false Fig. 4B, 4D cenpa-cenpc-1 3493630 N/A 3493635 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false N/A 3493634 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false flag tag 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 false physical association physical association false false false Fig. 6B cenpa-cenpc-2 3493630 N/A 3493635 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell flag tag 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 false N/A 3493634 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 6C Fig.7 Again the HA::CENP-A was immunoprecipitated and the IP complexes were analyzed by Western blotting with an anti-FLAG antibody. Results show that the C-terminal domain (aa 638–943) can be efficiently co-immunoprecipitated with CENP-A. Consistent results were obtained when the reciprocal experiment was performed. HA::CENP-C and FLAG::CENP-A constructs were cotransfected into HEK-293 cells, immunoprecipitated with an anti-HA antibody and FLAG-tagged proteins were detected (Figure 6C). cenpc-1 3493631 N/A 3493634 unspecified role unspecified role neutral component neutral component partially purified partially purified ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 2.0 physical association physical association false false false Fig. 5 cenpc-6 3493631 N/A 3493634 unspecified role unspecified role neutral component neutral component partially purified partially purified ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 3.0 physical association physical association false false false Fig. 5 cenpc-7 3493631 N/A 3493634 unspecified role unspecified role neutral component neutral component partially purified partially purified ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 4.0 physical association physical association false false false Fig. 5 cenpc-cenpc-1 3493632 N/A 3493635 unspecified role unspecified role bait bait subcellular prep subcellular preparation subcellular prep over-expressed over expressed level over-expressed ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false N/A 3493634 unspecified role unspecified role prey prey subcellular prep subcellular preparation subcellular prep physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association false false false Fig. 6A cenpa-cenpc-3 3493633 N/A 3493635 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false cgfp tag cgfp cGFP c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3493634 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed living cell living cell ngfp tag ngfp nGFP n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Fig. 8 Following the above described procedure we have performed a BiFC assay coexpressing N-GFP::HA::CENPCΔ890/943 and HA::CENPA::C-GFP. As shown in Figure 8DI we could observe only few green dots localizing at centromere positions (Figure 8DI–II) indicating a lowered efficiency level of the CENP-C/CENP-A interaction. In detail in this experiment only 25,6% of the green signal correspond to centromere positions (Figure 8G).