MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it http://mint.bio.uniroma2.it/mint anonymous-2014o-1 Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging human-tsa201 Homo sapiens subclone of human embryonic kidney 293 cell line that expresses simian virus 40 T antigen tsa201 Human embryonic kidney cell line subclone of 293 cell line that expresses simian virus 40 T antigen fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining jme1000@cam.ac.uk 2014 imex curation Antonio L.S., Stewart A.P., Varanda W.A., Michael Edwardson J. FEBS Lett. (0014-5793) Only protein-protein interactions anonymous-2014r-2 Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging human-tsa201 Homo sapiens subclone of human embryonic kidney 293 cell line that expresses simian virus 40 T antigen tsa201 Human embryonic kidney cell line subclone of 293 cell line that expresses simian virus 40 T antigen anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot jme1000@cam.ac.uk 2014 imex curation Antonio L.S., Stewart A.P., Varanda W.A., Michael Edwardson J. FEBS Lett. (0014-5793) Only protein-protein interactions p2rx2_rat P2X purinoceptor 2 P2rx2 ATP receptor Purinergic receptor protein protein rat Rattus norvegicus (Rat) Rat MVRRLARGCWSAFWDYETPKVIVVRNRRLGFVHRMVQLLILLYFVWYVFIVQKSYQDSETGPESSIITKVKGITMSEDKVWDVEEYVKPPEGGSVVSIITRIEVTPSQTLGTCPESMRVHSSTCHSDDDCIAGQLDMQGNGIRTGHCVPYYHGDSKTCEVSAWCPVEDGTSDNHFLGKMAPNFTILIKNSIHYPKFKFSKGNIASQKSDYLKHCTFDQDSDPYCPIFRLGFIVEKAGENFTELAHKGGVIGVIINWNCDLDLSESECNPKYSFRRLDPKYDPASSGYNFRFAKYYKINGTTTTRTLIKAYGIRIDVIVHGQAGKFSLIPTIINLATALTSIGVGSFLCDWILLTFMNKNKLYSHKKFDKVRTPKHPSSRWPVTLALVLGQIPPPPSHYSQDQPPSPPSGEGPTLGEGAELPLAVQSPRPCSISALTEQVVDTLGQHMGQRPPVPEPSQQDSTSTDPKGLAQL p2rx6_rat P2X purinoceptor 6 P2rx6 P2rxl1 ATP receptor P2XM Purinergic receptor Purinergic receptor P2X-like 1 protein protein rat Rattus norvegicus (Rat) Rat MQLQPAGTGSMASAVAAALVSWGFLDYKTEKYVMTRNCWVGISQRLLQLGVVVYVIGWALLAKKGYQEWDMDPQISVITKLKGVSVTQVKELEKRLWDVADFVRPSQGENVFFLVTNFLVTPAQVQGRCPEHPSVPLANCWADEDCPEGEMGTYSHGIKTGQCVAFNGTHRTCEIWSWCPVESSAVPRKPLLAQAKNFTLFIKNTVTFNKFNFSRTNALDTWDNTYFKYCLYDSLSSPYCPVFRIGDLVAMTGGDFEDLALLGGAVGINIHWDCNLDTKGSDCSPQYSFQLQERGYNFRTANYWWAASGVESRSLLKLYGIRFDILVTGQAGKFALIPTAITVGTGAAWLGMVTFLCDLLLLYVDREAGFYWRTKYEEARAPKATTNSA p2rx4_rat P2X purinoceptor 4 P2rx4 ATP receptor Purinergic receptor protein protein rat Rattus norvegicus (Rat) Rat MAGCCSVLGSFLFEYDTPRIVLIRSRKVGLMNRAVQLLILAYVIGWVFVWEKGYQETDSVVSSVTTKAKGVAVTNTSQLGFRIWDVADYVIPAQEENSLFIMTNMIVTVNQTQSTCPEIPDKTSICNSDADCTPGSVDTHSSGVATGRCVPFNESVKTCEVAAWCPVENDVGVPTPAFLKAAENFTLLVKNNIWYPKFNFSKRNILPNITTSYLKSCIYNAQTDPFCPIFRLGTIVEDAGHSFQEMAVEGGIMGIQIKWDCNLDRAASLCLPRYSFRRLDTRDLEHNVSPGYNFRFAKYYRDLAGKEQRTLTKAYGIRFDIIVFGKAGKFDIIPTMINVGSGLALLGVATVLCDVIVLYCMKKKYYYRDKKYKYVEDYEQGLSGEMNQ p2rx2-p2rx6 1535543 n/a P2X2 1535545 tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag 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 false n/a P2X6 1535546 anti tag immunost anti tag immunostaining anti tag immunost unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false colocalization colocalization false false false f1 Cells expressing various combinations of P2X receptor subunits (P2X2/P2X4, P2X2/P2X6, P2X4/P2X6 and P2X2/P2X4/P2X6) were fixed, permeabilized and incubated with appropriate primary antibodies, followed by fluorophore-conjugated secondary antibodies (Sigma). Cells were imaged by confocal laser scanning microscopy. As shown in Fig. 1, the immunostaining and transfection patterns coincided exactly, and the absence of a particular subunit led to a negative staining signal. The majority of co-transfected cells expressed each of the transfected subunits, and in all cases the proteins appeared to be present throughout the cell. p2rx2-p2rx6-1 1535543 n/a P2X6 1535546 anti tag immunost anti tag immunostaining anti tag immunost unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a P2X4 1535547 anti tag immunost anti tag immunostaining anti tag immunost unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false colocalization colocalization false false false f1 Cells expressing various combinations of P2X receptor subunits (P2X2/P2X4, P2X2/P2X6, P2X4/P2X6 and P2X2/P2X4/P2X6) were fixed, permeabilized and incubated with appropriate primary antibodies, followed by fluorophore-conjugated secondary antibodies (Sigma). Cells were imaged by confocal laser scanning microscopy. As shown in Fig. 1, the immunostaining and transfection patterns coincided exactly, and the absence of a particular subunit led to a negative staining signal. The majority of co-transfected cells expressed each of the transfected subunits, and in all cases the proteins appeared to be present throughout the cell. p2rx2-p2rx6-2 1535543 n/a P2X4 1535547 anti tag immunost anti tag immunostaining anti tag immunost unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a P2X2 1535545 tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag 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 false colocalization colocalization false false false f1 Cells expressing various combinations of P2X receptor subunits (P2X2/P2X4, P2X2/P2X6, P2X4/P2X6 and P2X2/P2X4/P2X6) were fixed, permeabilized and incubated with appropriate primary antibodies, followed by fluorophore-conjugated secondary antibodies (Sigma). Cells were imaged by confocal laser scanning microscopy. As shown in Fig. 1, the immunostaining and transfection patterns coincided exactly, and the absence of a particular subunit led to a negative staining signal. The majority of co-transfected cells expressed each of the transfected subunits, and in all cases the proteins appeared to be present throughout the cell. p2rx2-p2rx4 1535543 n/a P2X6 1535546 anti tag immunost anti tag immunostaining anti tag immunost unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell his tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a P2X2 1535545 tag fluorescence tag visualisation by fluorescence tag fluorescence unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag 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 false n/a P2X4 1535547 anti tag immunost anti tag immunostaining anti tag immunost unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false colocalization colocalization false false false f1 Cells expressing various combinations of P2X receptor subunits (P2X2/P2X4, P2X2/P2X6, P2X4/P2X6 and P2X2/P2X4/P2X6) were fixed, permeabilized and incubated with appropriate primary antibodies, followed by fluorophore-conjugated secondary antibodies (Sigma). Cells were imaged by confocal laser scanning microscopy. As shown in Fig. 1, the immunostaining and transfection patterns coincided exactly, and the absence of a particular subunit led to a negative staining signal. The majority of co-transfected cells expressed each of the transfected subunits, and in all cases the proteins appeared to be present throughout the cell. p2rx6-p2rx2 1535544 n/a P2X2 1535545 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a P2X6 1535546 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait 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 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false f2 p2rx6-p2rx2-1 1535544 n/a P2X2 1535545 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a P2X4 1535547 western blot western blot Immuno blot Immuno blot 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 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false physical association physical association false false false f2 p2rx6-p2rx2-2 1535544 n/a P2X4 1535547 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey partially purified partially purified ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a P2X2 1535545 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey partially purified partially purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a P2X6 1535546 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait partially purified partially purified flag tag 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 false association association false false false f2 A detergent extract of cells co-expressing His6-P2X2, P2X4-HA and FLAG-P2X6 was subjected to sequential anti-HA and anti-FLAG immunoaffinity chromatography steps (Fig. 2). The first (anti-HA) step would be expected capture any protein complex containing P2X4- HA, while the second (anti-FLAG) step would capture complexes containing both P2X4-HA and FLAG-P2X6. The immunoblots showed that this was indeed the case, and also indicated that His6-P2X2 was also present in the second eluate. The same result was obtained when the 8 order of the immunoaffinity chromatography steps was reversed. Fig. 2 also shows that when either FLAG-P2X6 or P2X4-HA was omitted, none of the three subunits appeared in the second eluate. These results indicate an intimate association between P2X2, P2X4 and P2X6. p2rx4-p2rx2 1535544 n/a P2X4 1535547 western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait partially purified partially purified ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false n/a P2X6 1535546 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey partially purified partially purified flag tag 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 false n/a P2X2 1535545 western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey partially purified partially purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false association association false false false f2 A detergent extract of cells co-expressing His6-P2X2, P2X4-HA and FLAG-P2X6 was subjected to sequential anti-HA and anti-FLAG immunoaffinity chromatography steps (Fig. 2). The first (anti-HA) step would be expected capture any protein complex containing P2X4- HA, while the second (anti-FLAG) step would capture complexes containing both P2X4-HA and FLAG-P2X6. The immunoblots showed that this was indeed the case, and also indicated that His6-P2X2 was also present in the second eluate. The same result was obtained when the 8 order of the immunoaffinity chromatography steps was reversed. Fig. 2 also shows that when either FLAG-P2X6 or P2X4-HA was omitted, none of the three subunits appeared in the second eluate. These results indicate an intimate association between P2X2, P2X4 and P2X6.