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 Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging. Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging. FEBS Lett. (0014-5793) 2014 imex curation Antonio L.S., Stewart A.P., Varanda W.A., Michael Edwardson J. jme1000@cam.ac.uk 11-Jun-2014: Contacted by IntAct-Help. Only protein-protein interactions 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 Accepted 2014-APR-28 by LUANA 2014 imex curation Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging. Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging. FEBS Lett. (0014-5793) 2014 imex curation Antonio L.S., Stewart A.P., Varanda W.A., Michael Edwardson J. jme1000@cam.ac.uk 11-Jun-2014: Contacted by IntAct-Help. Only protein-protein interactions 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 Accepted 2014-APR-28 by LUANA 2014 imex curation p2rx2_rat P2X purinoceptor 2 P2rx2 ATP receptor Purinergic receptor protein protein rat Rattus norvegicus (Rat) Rat MVRRLARGCWSAFWDYETPKVIVVRNRRLGFVHRMVQLLILLYFVWYVFIVQKSYQDSETGPESSIITKVKGITMSEDKVWDVEEYVKPPEGGSVVSIITRIEVTPSQTLGTCPESMRVHSSTCHSDDDCIAGQLDMQGNGIRTGHCVPYYHGDSKTCEVSAWCPVEDGTSDNHFLGKMAPNFTILIKNSIHYPKFKFSKGNIASQKSDYLKHCTFDQDSDPYCPIFRLGFIVEKAGENFTELAHKGGVIGVIINWNCDLDLSESECNPKYSFRRLDPKYDPASSGYNFRFAKYYKINGTTTTRTLIKAYGIRIDVIVHGQAGKFSLIPTIINLATALTSIGVGSFLCDWILLTFMNKNKLYSHKKFDKVRTPKHPSSRWPVTLALVLGQIPPPPSHYSQDQPPSPPSGEGPTLGEGAELPLAVQSPRPCSISALTEQVVDTLGQHMGQRPPVPEPSQQDSTSTDPKGLAQL AB53E09A2CADA6A2 p2rx4_rat P2X purinoceptor 4 P2rx4 ATP receptor Purinergic receptor protein protein rat Rattus norvegicus (Rat) Rat MAGCCSVLGSFLFEYDTPRIVLIRSRKVGLMNRAVQLLILAYVIGWVFVWEKGYQETDSVVSSVTTKAKGVAVTNTSQLGFRIWDVADYVIPAQEENSLFIMTNMIVTVNQTQSTCPEIPDKTSICNSDADCTPGSVDTHSSGVATGRCVPFNESVKTCEVAAWCPVENDVGVPTPAFLKAAENFTLLVKNNIWYPKFNFSKRNILPNITTSYLKSCIYNAQTDPFCPIFRLGTIVEDAGHSFQEMAVEGGIMGIQIKWDCNLDRAASLCLPRYSFRRLDTRDLEHNVSPGYNFRFAKYYRDLAGKEQRTLTKAYGIRFDIIVFGKAGKFDIIPTMINVGSGLALLGVATVLCDVIVLYCMKKKYYYRDKKYKYVEDYEQGLSGEMNQ DIP protein P51577 original sequence version: 79 C7B0FC9A94EF8D9A 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 1AE683A0191B6A0B p2rx2-p2rx6 2 P2X2 3 tag fluorescence tag visualisation by fluorescence tag fluorescence immunostaining immunostaining 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 P2X6 5 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining 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 colocalization colocalization 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 2 P2X6 5 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining 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 P2X4 4 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining 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 colocalization colocalization 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 2 P2X4 4 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining 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 P2X2 3 tag fluorescence tag visualisation by fluorescence tag fluorescence immunostaining immunostaining 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 colocalization colocalization 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 2 P2X6 5 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining 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 P2X2 3 tag fluorescence tag visualisation by fluorescence tag fluorescence immunostaining immunostaining 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 P2X4 4 anti tag immunost anti tag immunostaining anti tag immunost immunostaining immunostaining 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 colocalization colocalization 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 1 P2X2 3 western blot western blot Immuno blot Immuno blot 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 P2X6 5 western blot western blot Immuno blot Immuno blot 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 physical association physical association f2 p2rx6-p2rx2-1 1 P2X2 3 western blot western blot Immuno blot Immuno blot 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 P2X4 4 western blot western blot Immuno blot Immuno blot 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 physical association physical association f2 p2rx6-p2rx2-2 1 P2X4 4 western blot western blot Immuno blot Immuno blot 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 P2X2 3 western blot western blot Immuno blot Immuno blot 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 P2X6 5 western blot western blot Immuno blot Immuno blot 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 association association 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 1 P2X4 4 western blot western blot Immuno blot Immuno blot 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 P2X6 5 western blot western blot Immuno blot Immuno blot 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 P2X2 3 western blot western blot Immuno blot Immuno blot 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 association association 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.