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/ zamarin-2005-2 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 Zamarin D., García-Sastre A., Xiao X., Wang R., Palese P. Peter.Palese@mssm.edu PLoS pathogens 2005 Only protein-protein interactions imex curation imex curation zamarin-2005-3 in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Zamarin D., García-Sastre A., Xiao X., Wang R., Palese P. Peter.Palese@mssm.edu PLoS pathogens 2005 Only protein-protein interactions imex curation imex curation zamarin-2005-4 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. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Zamarin D., García-Sastre A., Xiao X., Wang R., Palese P. Peter.Palese@mssm.edu PLoS pathogens 2005 Only protein-protein interactions imex curation imex curation zamarin-2005-1 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. pull down pull down sequence tag sequence tag identification sequence tag Zamarin D., García-Sastre A., Xiao X., Wang R., Palese P. Beta Tubulin identified by mass spectrometry (Fig.5A) could not be mapped to a unique UniprotAc and was not captured. Peter.Palese@mssm.edu PLoS pathogens 2005 Only protein-protein interactions imex curation imex curation adt3_human ADP/ATP translocase 3 SLC25A6 ANT3 CDABP0051 Adenine nucleotide translocator 3 ADP,ATP carrier protein 3 Solute carrier family 25 member 6 ADP,ATP carrier protein, isoform T2 AAC3 protein protein human Homo sapiens Human MTEQAISFAKDFLAGGIAAAISKTAVAPIERVKLLLQVQHASKQIAADKQYKGIVDCIVRIPKEQGVLSFWRGNLANVIRYFPTQALNFAFKDKYKQIFLGGVDKHTQFWRYFAGNLASGGAAGATSLCFVYPLDFARTRLAADVGKSGTEREFRGLGDCLVKITKSDGIRGLYQGFSVSVQGIIIYRAAYFGVYDTAKGMLPDPKNTHIVVSWMIAQTVTAVAGVVSYPFDTVRRRMMMQSGRKGADIMYTGTVDCWRKIFRDEGGKAFFKGAWSNVLRGMGGAFVLVLYDELKKVI pb1f2_i34a1 Protein PB1-F2 PB1 protein protein i34a1 Influenza A virus (strain A/Puerto Rico/8/1934 H1N1) MGQEQDTPWILSTGHISTQKRQDGQQTPKLEHRNSTRLMGHCQKTMNQVVMPKQIVYWKQWLSLRNPILVFLKTRVLKRWRLFSKHE vdac1_human Voltage-dependent anion-selective channel protein 1 VDAC1 VDAC Outer mitochondrial membrane protein porin 1 Plasmalemmal porin Porin 31HL Porin 31HM protein protein human Homo sapiens Human MAVPPTYADLGKSARDVFTKGYGFGLIKLDLKTKSENGLEFTSSGSANTETTKVTGSLETKYRWTEYGLTFTEKWNTDNTLGTEITVEDQLARGLKLTFDSSFSPNTGKKNAKIKTGYKREHINLGCDMDFDIAGPSIRGALVLGYEGWLAGYQMNFETAKSRVTQSNFAVGYKTDEFQLHTNVNDGTEFGGSIYQKVNKKLETAVNLAWTAGNSNTRFGIAAKYQIDPDACFSAKVNNSSLIGLGYTQTLKPGIKLTLSALLDGKNVNAGGHKLGLGLEFQA pb1-slc25a6-1 3588694 N/A 3588698 unspecified role unspecified role prey prey 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 3588699 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed physical association physical association false false false Fig. 5B pb1-slc25a6-2 3588694 N/A 3588700 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed 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 3588699 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed physical association physical association false false false Fig. 5B pb1-vdac1-1 3588694 N/A 3588700 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed 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 3588699 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association false false false Fig. 5E Immunoprecipitation experiments with anti-PB1-F2 polyclonal serum revealed that the PB1-F2 protein interacts with ANT3 and VDAC1 during the viral infection weak interaction pb1-vdac1-2 3588694 N/A 3588698 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed 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 3588699 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association false false false Fig. 5E Immunoprecipitation experiments with anti-PB1-F2 polyclonal serum revealed that the PB1-F2 protein interacts with ANT3 and VDAC1 during the viral infection pb1-slc25a6-3 3588695 N/A 3588698 unspecified role unspecified role prey prey cell lysate cell lysate in vitro translated in vitro translated protein in vitro translated 35s radiolabel 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes N/A 3588699 unspecified role unspecified role bait bait purified purified gst tag 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 ecoli Escherichia coli (strain K12) physical association physical association false false false Fig. 5D pb1-slc25a6-4 3588695 N/A 3588700 unspecified role unspecified role prey prey cell lysate cell lysate in vitro translated in vitro translated protein in vitro translated 35s radiolabel 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes N/A 3588699 unspecified role unspecified role bait bait purified purified gst tag 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 ecoli Escherichia coli (strain K12) physical association physical association false false false Fig. 5D pb1-slc25a6-5 3588696 N/A 3588699 unspecified role unspecified role bait bait 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 gfp tag 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 false c-terminal_region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false N/A 3588698 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed 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. 6C pb1-slc25a6-6 3588696 N/A 3588699 unspecified role unspecified role bait bait 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 gfp tag 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 false c-terminal_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n-terminal_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3588700 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed 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. 6D psf2-1 3588697 N/A 3588698 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level N/A 3588699 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed gst tag 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 physical association physical association false false false Fig. 5A Mass spectrometry analysis of the proteins identified the 36-kDa band as the mitochondrial ANT3, the 55-kDa band as beta tubulin, and the 80-kDa band as cytokeratin. The latter was likely a contaminant of the protein preparation. While interaction of PB1-F2 with tubulin may be important for PB1-F2-induced mitochondrial disorganization.