Structured Review

Millipore action against p2x1
Action Against P2x1, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/action against p2x1/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
action against p2x1 - by Bioz Stars, 2024-07
86/100 stars

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p2x1 7r  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier
Bioz Manufacturer Symbol Thermo Fisher manufactures this product  
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    Thermo Fisher p2x1 7r
    P2x1 7r, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2x1 7r/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2x1 7r - by Bioz Stars, 2024-07
    86/100 stars

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    Structured Review

    Promega detergent purified p2x1 receptor
    A) Cryo-EM map of the ATP-bound <t>P2X1</t> receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.
    Detergent Purified P2x1 Receptor, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/detergent purified p2x1 receptor/product/Promega
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    detergent purified p2x1 receptor - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "Structural insights into the human P2X1 receptor and ligand interactions"

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    Journal: bioRxiv

    doi: 10.1101/2024.04.04.588192

    A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.
    Figure Legend Snippet: A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.

    Techniques Used: Cryo-EM Sample Prep

    Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).
    Figure Legend Snippet: Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).

    Techniques Used:

    A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.
    Figure Legend Snippet: A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

    Techniques Used: Binding Assay


    Figure Legend Snippet:

    Techniques Used: Derivative Assay, Expressing, Residue

    A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.
    Figure Legend Snippet: A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Techniques Used: Binding Assay, Labeling, Expressing, Residue, Sequencing


    Figure Legend Snippet:

    Techniques Used: Derivative Assay, Expressing, Residue

    A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.
    Figure Legend Snippet: A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Techniques Used: Binding Assay, Mutagenesis, Expressing, Residue, Sequencing

    Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.
    Figure Legend Snippet: Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.

    Techniques Used: Binding Assay

    Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.
    Figure Legend Snippet: Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.

    Techniques Used: Binding Assay

    trimeric p2x1 receptor  (Danaher Inc)


    Bioz Verified Symbol Danaher Inc is a verified supplier
    Bioz Manufacturer Symbol Danaher Inc manufactures this product  
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    Structured Review

    Danaher Inc trimeric p2x1 receptor
    A) Cryo-EM map of the ATP-bound <t>P2X1</t> receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.
    Trimeric P2x1 Receptor, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trimeric p2x1 receptor/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trimeric p2x1 receptor - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "Structural insights into the human P2X1 receptor and ligand interactions"

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    Journal: bioRxiv

    doi: 10.1101/2024.04.04.588192

    A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.
    Figure Legend Snippet: A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.

    Techniques Used: Cryo-EM Sample Prep

    Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).
    Figure Legend Snippet: Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).

    Techniques Used:

    A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.
    Figure Legend Snippet: A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

    Techniques Used: Binding Assay


    Figure Legend Snippet:

    Techniques Used: Derivative Assay, Expressing, Residue

    A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.
    Figure Legend Snippet: A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Techniques Used: Binding Assay, Labeling, Expressing, Residue, Sequencing


    Figure Legend Snippet:

    Techniques Used: Derivative Assay, Expressing, Residue

    A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.
    Figure Legend Snippet: A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Techniques Used: Binding Assay, Mutagenesis, Expressing, Residue, Sequencing

    Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.
    Figure Legend Snippet: Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.

    Techniques Used: Binding Assay

    Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.
    Figure Legend Snippet: Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.

    Techniques Used: Binding Assay

    human p2x1  (OriGene)


    Bioz Verified Symbol OriGene is a verified supplier
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    Structured Review

    OriGene human p2x1
    Human P2x1, supplied by OriGene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human p2x1/product/OriGene
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human p2x1 - by Bioz Stars, 2024-07
    86/100 stars

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    anti p2x1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
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  • 86

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    Alomone Labs anti p2x1
    Mast cell P2X receptor expression and modulation by IL-33. ( A – C ) Representative histogram indicating <t>P2X1</t> ( A ), P2X4 ( B ), and P2X7 ( C ) expression upon treatment with media (5 or 50 ng/mL IL-33 for 24 h). P2X1, P2X4, and P2X7 were stained and analysed by flow cytometry. The dotted lines indicate cell staining with isotype control antibodies. ( D – F ) IL-33-treated or untreated MCs were stained for P2X1 ( n = 2), P2X4 ( n = 5), and P2X7 ( n = 4); the geometric mean of fluorescence intensity (GMFI) was normalized to the negative control (untreated samples). Data are displayed as mean ± SEM. Statistical differences are indicated by * p < 0.05 and ** p < 0.01, *** p < 0.001 (one-way ANOVA with Šídák’s post hoc test).
    Anti P2x1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    anti p2x1 - by Bioz Stars, 2024-07
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    Images

    1) Product Images from "P2X7 Receptor-Induced Human Mast Cell Degranulation Is Enhanced by Interleukin 33"

    Article Title: P2X7 Receptor-Induced Human Mast Cell Degranulation Is Enhanced by Interleukin 33

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms25031730

    Mast cell P2X receptor expression and modulation by IL-33. ( A – C ) Representative histogram indicating P2X1 ( A ), P2X4 ( B ), and P2X7 ( C ) expression upon treatment with media (5 or 50 ng/mL IL-33 for 24 h). P2X1, P2X4, and P2X7 were stained and analysed by flow cytometry. The dotted lines indicate cell staining with isotype control antibodies. ( D – F ) IL-33-treated or untreated MCs were stained for P2X1 ( n = 2), P2X4 ( n = 5), and P2X7 ( n = 4); the geometric mean of fluorescence intensity (GMFI) was normalized to the negative control (untreated samples). Data are displayed as mean ± SEM. Statistical differences are indicated by * p < 0.05 and ** p < 0.01, *** p < 0.001 (one-way ANOVA with Šídák’s post hoc test).
    Figure Legend Snippet: Mast cell P2X receptor expression and modulation by IL-33. ( A – C ) Representative histogram indicating P2X1 ( A ), P2X4 ( B ), and P2X7 ( C ) expression upon treatment with media (5 or 50 ng/mL IL-33 for 24 h). P2X1, P2X4, and P2X7 were stained and analysed by flow cytometry. The dotted lines indicate cell staining with isotype control antibodies. ( D – F ) IL-33-treated or untreated MCs were stained for P2X1 ( n = 2), P2X4 ( n = 5), and P2X7 ( n = 4); the geometric mean of fluorescence intensity (GMFI) was normalized to the negative control (untreated samples). Data are displayed as mean ± SEM. Statistical differences are indicated by * p < 0.05 and ** p < 0.01, *** p < 0.001 (one-way ANOVA with Šídák’s post hoc test).

    Techniques Used: Expressing, Staining, Flow Cytometry, Fluorescence, Negative Control

    The use of P2X1 and P2X4 receptors, alone or in combination, does not affect MC degranulation. MCs were left untreated or incubated with 5 ng/mL IL-33 for 24 h. ( A ) The MCs were exposed to NF449 (P2X1 inhibitor) and 5BDBD (P2X4 inhibitor) for 15 min before ATP stimulation at the concentrations indicated ( n = 3 separate experiments). A statistical analysis showed no significance between the control, stimulated, and IL-33-primed cells. ( B ) The MCs were exposed to NF449 (P2X1 inhibitor), 5BDBD (P2X4 inhibitor), and A438079 (P2X7 inhibitor) alone or in combination for 15 min before 1000 µM ATP stimulation at the concentrations indicated ( n = 3 separate experiments using different MC cultures). Statistical analysis showed no significant difference between the untreated controls and IL-33-primed cells. Data are presented as mean ± SEM. Statistical differences are indicated; * p < 0.05 (one-way ANOVA with Sidak’s post hoc test).
    Figure Legend Snippet: The use of P2X1 and P2X4 receptors, alone or in combination, does not affect MC degranulation. MCs were left untreated or incubated with 5 ng/mL IL-33 for 24 h. ( A ) The MCs were exposed to NF449 (P2X1 inhibitor) and 5BDBD (P2X4 inhibitor) for 15 min before ATP stimulation at the concentrations indicated ( n = 3 separate experiments). A statistical analysis showed no significance between the control, stimulated, and IL-33-primed cells. ( B ) The MCs were exposed to NF449 (P2X1 inhibitor), 5BDBD (P2X4 inhibitor), and A438079 (P2X7 inhibitor) alone or in combination for 15 min before 1000 µM ATP stimulation at the concentrations indicated ( n = 3 separate experiments using different MC cultures). Statistical analysis showed no significant difference between the untreated controls and IL-33-primed cells. Data are presented as mean ± SEM. Statistical differences are indicated; * p < 0.05 (one-way ANOVA with Sidak’s post hoc test).

    Techniques Used: Incubation

    rabbit anti p2x1 antibody  (Alomone Labs)


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    Alomone Labs rabbit anti p2x1 antibody
    Rabbit Anti P2x1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p2x1  (Tocris)


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    Tocris p2x1
    Purinergic receptor antagonists.
    P2x1, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Besnoitia besnoiti -induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling"

    Article Title: Besnoitia besnoiti -induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2023.1244068

    Purinergic receptor antagonists.
    Figure Legend Snippet: Purinergic receptor antagonists.

    Techniques Used:

    Effects of purinergic receptor antagonist pre-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were pre-treated for 10 min with increasing concentrations of MRS2179, MRS2578, NF449, 5-BDBD, and AZ10606120 (0.1-100 µM) targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively, and then exposed to B. besnoiti tachyzoites for 4 (h) Thereafter, cell-free (A, C, E, G, I) and anchored (B, D, F, H, J) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p -values were calculated by Kruskal-Wallis test followed by Dunn´s multiple comparison test ( n =6). * p < 0,05; ** p < 0,01.
    Figure Legend Snippet: Effects of purinergic receptor antagonist pre-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were pre-treated for 10 min with increasing concentrations of MRS2179, MRS2578, NF449, 5-BDBD, and AZ10606120 (0.1-100 µM) targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively, and then exposed to B. besnoiti tachyzoites for 4 (h) Thereafter, cell-free (A, C, E, G, I) and anchored (B, D, F, H, J) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p -values were calculated by Kruskal-Wallis test followed by Dunn´s multiple comparison test ( n =6). * p < 0,05; ** p < 0,01.

    Techniques Used: Derivative Assay, Fluorescence, Comparison

    Effects of purinergic receptor antagonist post-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were first exposed to B. besnoiti tachyzoites for 40 min and then treated with 100 µM of MRS2179, MRS2578, NF449, and 5-BDBD targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively. After 4 h of incubation, cell-free (A, C) and anchored (B, D) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p -values were calculated by an ordinary one-way ANOVA with Dunnett´s multiple comparison analysis. ( n =3). * p < 0,05; ** p < 0,01; *** p < 0,001.
    Figure Legend Snippet: Effects of purinergic receptor antagonist post-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were first exposed to B. besnoiti tachyzoites for 40 min and then treated with 100 µM of MRS2179, MRS2578, NF449, and 5-BDBD targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively. After 4 h of incubation, cell-free (A, C) and anchored (B, D) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p -values were calculated by an ordinary one-way ANOVA with Dunnett´s multiple comparison analysis. ( n =3). * p < 0,05; ** p < 0,01; *** p < 0,001.

    Techniques Used: Incubation, Derivative Assay, Fluorescence, Comparison

    B. besnoiti tachyzoite-induced clustering of bovine PMN depends on P2X1-based purinergic signaling. PMN were co-cultured with tachyzoites for 4 h in the presence or absence of NF449 (100 µM). (A) Exemplary illustrations of tachyzoite-PMN co-cultures stained for DNA (DAPI, blue) and parasite stages (red). (B) DANA-based quantification of cluster formation. All values are presented as mean ± SD, and p -values were calculated using one-way ANOVA followed by Tukey´s multiple comparisons. ( n = 4). * p < 0,05; ** p < 0,01.
    Figure Legend Snippet: B. besnoiti tachyzoite-induced clustering of bovine PMN depends on P2X1-based purinergic signaling. PMN were co-cultured with tachyzoites for 4 h in the presence or absence of NF449 (100 µM). (A) Exemplary illustrations of tachyzoite-PMN co-cultures stained for DNA (DAPI, blue) and parasite stages (red). (B) DANA-based quantification of cluster formation. All values are presented as mean ± SD, and p -values were calculated using one-way ANOVA followed by Tukey´s multiple comparisons. ( n = 4). * p < 0,05; ** p < 0,01.

    Techniques Used: Cell Culture, Staining

    p2x1 receptor  (MedChemExpress)


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    MedChemExpress p2x1 receptor
    P2x1 Receptor, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jumpstart Fertility rat p2x1 7
    Rat P2x1 7, supplied by Jumpstart Fertility, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A) Cryo-EM map of the ATP-bound <t>P2X1</t> receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.
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    A) Cryo-EM map of the ATP-bound <t>P2X1</t> receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.
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    Mast cell P2X receptor expression and modulation by IL-33. ( A – C ) Representative histogram indicating <t>P2X1</t> ( A ), P2X4 ( B ), and P2X7 ( C ) expression upon treatment with media (5 or 50 ng/mL IL-33 for 24 h). P2X1, P2X4, and P2X7 were stained and analysed by flow cytometry. The dotted lines indicate cell staining with isotype control antibodies. ( D – F ) IL-33-treated or untreated MCs were stained for P2X1 ( n = 2), P2X4 ( n = 5), and P2X7 ( n = 4); the geometric mean of fluorescence intensity (GMFI) was normalized to the negative control (untreated samples). Data are displayed as mean ± SEM. Statistical differences are indicated by * p < 0.05 and ** p < 0.01, *** p < 0.001 (one-way ANOVA with Šídák’s post hoc test).
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    Mast cell P2X receptor expression and modulation by IL-33. ( A – C ) Representative histogram indicating <t>P2X1</t> ( A ), P2X4 ( B ), and P2X7 ( C ) expression upon treatment with media (5 or 50 ng/mL IL-33 for 24 h). P2X1, P2X4, and P2X7 were stained and analysed by flow cytometry. The dotted lines indicate cell staining with isotype control antibodies. ( D – F ) IL-33-treated or untreated MCs were stained for P2X1 ( n = 2), P2X4 ( n = 5), and P2X7 ( n = 4); the geometric mean of fluorescence intensity (GMFI) was normalized to the negative control (untreated samples). Data are displayed as mean ± SEM. Statistical differences are indicated by * p < 0.05 and ** p < 0.01, *** p < 0.001 (one-way ANOVA with Šídák’s post hoc test).
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    Purinergic receptor antagonists.
    P2x1, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.

    Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

    Techniques: Cryo-EM Sample Prep

    Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).

    Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

    Techniques:

    A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

    Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

    Techniques: Binding Assay

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet:

    Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

    Techniques: Derivative Assay, Expressing, Residue

    A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

    Techniques: Binding Assay, Labeling, Expressing, Residue, Sequencing

    A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

    Techniques: Binding Assay, Mutagenesis, Expressing, Residue, Sequencing

    Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.

    Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

    Techniques: Binding Assay

    Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.

    Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

    Techniques: Binding Assay

    A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.

    Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

    Techniques: Cryo-EM Sample Prep

    Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).

    Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

    Techniques:

    A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

    Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

    Techniques: Binding Assay

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet:

    Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

    Techniques: Derivative Assay, Expressing, Residue

    A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

    Techniques: Binding Assay, Labeling, Expressing, Residue, Sequencing

    A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

    Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

    Techniques: Binding Assay, Mutagenesis, Expressing, Residue, Sequencing

    Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.

    Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

    Techniques: Binding Assay

    Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.

    Journal: bioRxiv

    Article Title: Structural insights into the human P2X1 receptor and ligand interactions

    doi: 10.1101/2024.04.04.588192

    Figure Lengend Snippet: Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.

    Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

    Techniques: Binding Assay

    Mast cell P2X receptor expression and modulation by IL-33. ( A – C ) Representative histogram indicating P2X1 ( A ), P2X4 ( B ), and P2X7 ( C ) expression upon treatment with media (5 or 50 ng/mL IL-33 for 24 h). P2X1, P2X4, and P2X7 were stained and analysed by flow cytometry. The dotted lines indicate cell staining with isotype control antibodies. ( D – F ) IL-33-treated or untreated MCs were stained for P2X1 ( n = 2), P2X4 ( n = 5), and P2X7 ( n = 4); the geometric mean of fluorescence intensity (GMFI) was normalized to the negative control (untreated samples). Data are displayed as mean ± SEM. Statistical differences are indicated by * p < 0.05 and ** p < 0.01, *** p < 0.001 (one-way ANOVA with Šídák’s post hoc test).

    Journal: International Journal of Molecular Sciences

    Article Title: P2X7 Receptor-Induced Human Mast Cell Degranulation Is Enhanced by Interleukin 33

    doi: 10.3390/ijms25031730

    Figure Lengend Snippet: Mast cell P2X receptor expression and modulation by IL-33. ( A – C ) Representative histogram indicating P2X1 ( A ), P2X4 ( B ), and P2X7 ( C ) expression upon treatment with media (5 or 50 ng/mL IL-33 for 24 h). P2X1, P2X4, and P2X7 were stained and analysed by flow cytometry. The dotted lines indicate cell staining with isotype control antibodies. ( D – F ) IL-33-treated or untreated MCs were stained for P2X1 ( n = 2), P2X4 ( n = 5), and P2X7 ( n = 4); the geometric mean of fluorescence intensity (GMFI) was normalized to the negative control (untreated samples). Data are displayed as mean ± SEM. Statistical differences are indicated by * p < 0.05 and ** p < 0.01, *** p < 0.001 (one-way ANOVA with Šídák’s post hoc test).

    Article Snippet: The cells were then washed in FACS buffer (PBS, 2% v / v FCS, 2 mM EDTA) and incubated with either anti-P2X1 (1 mg/mL) (Cat# APR-022; isotype rabbit IgG1), anti-P2X4 (1 mg/mL) (Cat# APR-024; isotype rabbit IgG1), or anti-P2X7 (1 mg/mL) (Cat# APR-008; isotype rabbit IgG1; Alomone labs, Jerusalem, Israel) primary antibodies, together with 5 µg/mL of Fc block.

    Techniques: Expressing, Staining, Flow Cytometry, Fluorescence, Negative Control

    The use of P2X1 and P2X4 receptors, alone or in combination, does not affect MC degranulation. MCs were left untreated or incubated with 5 ng/mL IL-33 for 24 h. ( A ) The MCs were exposed to NF449 (P2X1 inhibitor) and 5BDBD (P2X4 inhibitor) for 15 min before ATP stimulation at the concentrations indicated ( n = 3 separate experiments). A statistical analysis showed no significance between the control, stimulated, and IL-33-primed cells. ( B ) The MCs were exposed to NF449 (P2X1 inhibitor), 5BDBD (P2X4 inhibitor), and A438079 (P2X7 inhibitor) alone or in combination for 15 min before 1000 µM ATP stimulation at the concentrations indicated ( n = 3 separate experiments using different MC cultures). Statistical analysis showed no significant difference between the untreated controls and IL-33-primed cells. Data are presented as mean ± SEM. Statistical differences are indicated; * p < 0.05 (one-way ANOVA with Sidak’s post hoc test).

    Journal: International Journal of Molecular Sciences

    Article Title: P2X7 Receptor-Induced Human Mast Cell Degranulation Is Enhanced by Interleukin 33

    doi: 10.3390/ijms25031730

    Figure Lengend Snippet: The use of P2X1 and P2X4 receptors, alone or in combination, does not affect MC degranulation. MCs were left untreated or incubated with 5 ng/mL IL-33 for 24 h. ( A ) The MCs were exposed to NF449 (P2X1 inhibitor) and 5BDBD (P2X4 inhibitor) for 15 min before ATP stimulation at the concentrations indicated ( n = 3 separate experiments). A statistical analysis showed no significance between the control, stimulated, and IL-33-primed cells. ( B ) The MCs were exposed to NF449 (P2X1 inhibitor), 5BDBD (P2X4 inhibitor), and A438079 (P2X7 inhibitor) alone or in combination for 15 min before 1000 µM ATP stimulation at the concentrations indicated ( n = 3 separate experiments using different MC cultures). Statistical analysis showed no significant difference between the untreated controls and IL-33-primed cells. Data are presented as mean ± SEM. Statistical differences are indicated; * p < 0.05 (one-way ANOVA with Sidak’s post hoc test).

    Article Snippet: The cells were then washed in FACS buffer (PBS, 2% v / v FCS, 2 mM EDTA) and incubated with either anti-P2X1 (1 mg/mL) (Cat# APR-022; isotype rabbit IgG1), anti-P2X4 (1 mg/mL) (Cat# APR-024; isotype rabbit IgG1), or anti-P2X7 (1 mg/mL) (Cat# APR-008; isotype rabbit IgG1; Alomone labs, Jerusalem, Israel) primary antibodies, together with 5 µg/mL of Fc block.

    Techniques: Incubation

    Purinergic receptor antagonists.

    Journal: Frontiers in Immunology

    Article Title: Besnoitia besnoiti -induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling

    doi: 10.3389/fimmu.2023.1244068

    Figure Lengend Snippet: Purinergic receptor antagonists.

    Article Snippet: P2X1 , NF449 , 1391 (Tocris).

    Techniques:

    Effects of purinergic receptor antagonist pre-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were pre-treated for 10 min with increasing concentrations of MRS2179, MRS2578, NF449, 5-BDBD, and AZ10606120 (0.1-100 µM) targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively, and then exposed to B. besnoiti tachyzoites for 4 (h) Thereafter, cell-free (A, C, E, G, I) and anchored (B, D, F, H, J) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p -values were calculated by Kruskal-Wallis test followed by Dunn´s multiple comparison test ( n =6). * p < 0,05; ** p < 0,01.

    Journal: Frontiers in Immunology

    Article Title: Besnoitia besnoiti -induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling

    doi: 10.3389/fimmu.2023.1244068

    Figure Lengend Snippet: Effects of purinergic receptor antagonist pre-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were pre-treated for 10 min with increasing concentrations of MRS2179, MRS2578, NF449, 5-BDBD, and AZ10606120 (0.1-100 µM) targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively, and then exposed to B. besnoiti tachyzoites for 4 (h) Thereafter, cell-free (A, C, E, G, I) and anchored (B, D, F, H, J) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p -values were calculated by Kruskal-Wallis test followed by Dunn´s multiple comparison test ( n =6). * p < 0,05; ** p < 0,01.

    Article Snippet: P2X1 , NF449 , 1391 (Tocris).

    Techniques: Derivative Assay, Fluorescence, Comparison

    Effects of purinergic receptor antagonist post-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were first exposed to B. besnoiti tachyzoites for 40 min and then treated with 100 µM of MRS2179, MRS2578, NF449, and 5-BDBD targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively. After 4 h of incubation, cell-free (A, C) and anchored (B, D) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p -values were calculated by an ordinary one-way ANOVA with Dunnett´s multiple comparison analysis. ( n =3). * p < 0,05; ** p < 0,01; *** p < 0,001.

    Journal: Frontiers in Immunology

    Article Title: Besnoitia besnoiti -induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling

    doi: 10.3389/fimmu.2023.1244068

    Figure Lengend Snippet: Effects of purinergic receptor antagonist post-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were first exposed to B. besnoiti tachyzoites for 40 min and then treated with 100 µM of MRS2179, MRS2578, NF449, and 5-BDBD targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively. After 4 h of incubation, cell-free (A, C) and anchored (B, D) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p -values were calculated by an ordinary one-way ANOVA with Dunnett´s multiple comparison analysis. ( n =3). * p < 0,05; ** p < 0,01; *** p < 0,001.

    Article Snippet: P2X1 , NF449 , 1391 (Tocris).

    Techniques: Incubation, Derivative Assay, Fluorescence, Comparison

    B. besnoiti tachyzoite-induced clustering of bovine PMN depends on P2X1-based purinergic signaling. PMN were co-cultured with tachyzoites for 4 h in the presence or absence of NF449 (100 µM). (A) Exemplary illustrations of tachyzoite-PMN co-cultures stained for DNA (DAPI, blue) and parasite stages (red). (B) DANA-based quantification of cluster formation. All values are presented as mean ± SD, and p -values were calculated using one-way ANOVA followed by Tukey´s multiple comparisons. ( n = 4). * p < 0,05; ** p < 0,01.

    Journal: Frontiers in Immunology

    Article Title: Besnoitia besnoiti -induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling

    doi: 10.3389/fimmu.2023.1244068

    Figure Lengend Snippet: B. besnoiti tachyzoite-induced clustering of bovine PMN depends on P2X1-based purinergic signaling. PMN were co-cultured with tachyzoites for 4 h in the presence or absence of NF449 (100 µM). (A) Exemplary illustrations of tachyzoite-PMN co-cultures stained for DNA (DAPI, blue) and parasite stages (red). (B) DANA-based quantification of cluster formation. All values are presented as mean ± SD, and p -values were calculated using one-way ANOVA followed by Tukey´s multiple comparisons. ( n = 4). * p < 0,05; ** p < 0,01.

    Article Snippet: P2X1 , NF449 , 1391 (Tocris).

    Techniques: Cell Culture, Staining