p2x5  (Alomone Labs)


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    Alomone Labs p2x5
    A role for peripheral GDNF signaling in the development of ischemic myalgia. After I/R, injured muscles release a variety of metabolites that include ATP, lactate and protons that stimulate group III and IV muscle afferents. Simultaneously, GDNF is being release in the injured tissues and this induces a signaling cascade in primary sensory neurons through its receptor GFRα1 that involves increased function of CREB/ CREB binding protein (CBP), which in turn modulates the observed overexpression of ATP sensitive channel <t>P2X5</t> and to a lesser degree, ASIC3. This molecular change at the DRG level in turn modulates the appearance of pain-related behaviors such as paw guarding, mechanical hypersensitivity and muscle weakness. In addition, this pathway also affects the exercise pressor reflex after injury. Targeting the overexpression of P2X5 or GFRα1 in sensory neurons or the increased GDNF levels within the muscle appears to inhibit many of these ischemic myalgia-like phenomena.
    P2x5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2x5/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2x5 - by Bioz Stars, 2022-07
    91/100 stars

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    1) Product Images from "A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes"

    Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

    Journal: bioRxiv

    doi: 10.1101/627521

    A role for peripheral GDNF signaling in the development of ischemic myalgia. After I/R, injured muscles release a variety of metabolites that include ATP, lactate and protons that stimulate group III and IV muscle afferents. Simultaneously, GDNF is being release in the injured tissues and this induces a signaling cascade in primary sensory neurons through its receptor GFRα1 that involves increased function of CREB/ CREB binding protein (CBP), which in turn modulates the observed overexpression of ATP sensitive channel P2X5 and to a lesser degree, ASIC3. This molecular change at the DRG level in turn modulates the appearance of pain-related behaviors such as paw guarding, mechanical hypersensitivity and muscle weakness. In addition, this pathway also affects the exercise pressor reflex after injury. Targeting the overexpression of P2X5 or GFRα1 in sensory neurons or the increased GDNF levels within the muscle appears to inhibit many of these ischemic myalgia-like phenomena.
    Figure Legend Snippet: A role for peripheral GDNF signaling in the development of ischemic myalgia. After I/R, injured muscles release a variety of metabolites that include ATP, lactate and protons that stimulate group III and IV muscle afferents. Simultaneously, GDNF is being release in the injured tissues and this induces a signaling cascade in primary sensory neurons through its receptor GFRα1 that involves increased function of CREB/ CREB binding protein (CBP), which in turn modulates the observed overexpression of ATP sensitive channel P2X5 and to a lesser degree, ASIC3. This molecular change at the DRG level in turn modulates the appearance of pain-related behaviors such as paw guarding, mechanical hypersensitivity and muscle weakness. In addition, this pathway also affects the exercise pressor reflex after injury. Targeting the overexpression of P2X5 or GFRα1 in sensory neurons or the increased GDNF levels within the muscle appears to inhibit many of these ischemic myalgia-like phenomena.

    Techniques Used: Binding Assay, Over Expression

    Disruption of the CBP/CREB transcription factor complex regulates I/R-related pain-like behaviors, EPRs and P2X5 expression. A . pCREB and CBP are increased after I/R compared to naïve as assessed with western blotting. Penα1 injection however partially blocked the I/R-induced upregulation of pCREB and fully inhibited the upregulation of CBP in the DRGs. B. Paw guarding is increased 1d after I/R in vehicle treated (0.1% DMSO) animals and this is completely prevented by treatment with the CREB-CBP interaction antagonist xx-650-23. Mechanical withdrawal thresholds to VFH stimulation (C) and muscle squeezing (D) are significantly decreased after I/R+0.1%DMSO and I/R+xx-650-23. However, both of these I/R-induced behavioral effects were significantly blunted in xx-650-23 treated animals. E . Decreased grip strength observed 1d after I/R+0.1%DMSO was partially rescued by xx-650-23. F. Exercise pressor reflexes (reflected by changes in MAP after forced running) exacerbated by I/R were also blocked by disruption of the CBP/CREB complex G. xx-650-23 significantly reduced the levels of P2X5 in the DRGs of mice with I/R compared to I/R-injured animals treated with vehicle (0.1% DMSO). (A: n=4-7 per group, *p
    Figure Legend Snippet: Disruption of the CBP/CREB transcription factor complex regulates I/R-related pain-like behaviors, EPRs and P2X5 expression. A . pCREB and CBP are increased after I/R compared to naïve as assessed with western blotting. Penα1 injection however partially blocked the I/R-induced upregulation of pCREB and fully inhibited the upregulation of CBP in the DRGs. B. Paw guarding is increased 1d after I/R in vehicle treated (0.1% DMSO) animals and this is completely prevented by treatment with the CREB-CBP interaction antagonist xx-650-23. Mechanical withdrawal thresholds to VFH stimulation (C) and muscle squeezing (D) are significantly decreased after I/R+0.1%DMSO and I/R+xx-650-23. However, both of these I/R-induced behavioral effects were significantly blunted in xx-650-23 treated animals. E . Decreased grip strength observed 1d after I/R+0.1%DMSO was partially rescued by xx-650-23. F. Exercise pressor reflexes (reflected by changes in MAP after forced running) exacerbated by I/R were also blocked by disruption of the CBP/CREB complex G. xx-650-23 significantly reduced the levels of P2X5 in the DRGs of mice with I/R compared to I/R-injured animals treated with vehicle (0.1% DMSO). (A: n=4-7 per group, *p

    Techniques Used: Expressing, Western Blot, Injection, Mouse Assay

    I/R causes and increase in the total number of cells that are positive for GFRα1 and P2X5. A . Representative images of DRG sections immunostained for GFRα1 and P2X5. Yellow arrows mark GFRα1 negative cells and white arrows mark positive cells. B-D . After I/R there was a significant increase in the total number of cells immunopositive for both GFRα1 and P2X5 (B) as well as total cells positive for either GFRα1 (C) or P2X5 (D) . The increase in immunopositive cells was prevented in Penα1+I/R. (n=3 per group, *p
    Figure Legend Snippet: I/R causes and increase in the total number of cells that are positive for GFRα1 and P2X5. A . Representative images of DRG sections immunostained for GFRα1 and P2X5. Yellow arrows mark GFRα1 negative cells and white arrows mark positive cells. B-D . After I/R there was a significant increase in the total number of cells immunopositive for both GFRα1 and P2X5 (B) as well as total cells positive for either GFRα1 (C) or P2X5 (D) . The increase in immunopositive cells was prevented in Penα1+I/R. (n=3 per group, *p

    Techniques Used:

    Selective knockdown of P2X5 in primary muscle afferents prevents the development of pain-related behaviors and exacerbated EPRs after I/R. A . P2X5 is increased in DRGs 1d after I/R or PenCON+I/R and this is prevented by PenX5 injection in mice with I/R (n=3 per group). B. Paw guarding is increased 1d after I/R and PenCON+I/R compared to sham controls, and this is partially prevented in PenX5+I/R animals. C. Mechanical withdrawal thresholds to von Frey hair (VFH) stimulation are decreased after I/R and PenCON+I/R and this is partially recued in the PenX5+I/R group. D. Mechanical withdrawal thresholds to muscle squeezing are decreased after I/R, PenCON+I/R and PenX5+I/R. E. PenX5 injection in mice with I/R also partially inhibited I/R-related grip strength deficits observed in I/R alone or PenCON+I/R groups. F. A significant increase in mean arterial pressure (MAP) after exercise was observed in injured mice (I/R and PenCON+I/R), but this was not observed in the PenX5+I/R group. (D-E n=12 per group. x p
    Figure Legend Snippet: Selective knockdown of P2X5 in primary muscle afferents prevents the development of pain-related behaviors and exacerbated EPRs after I/R. A . P2X5 is increased in DRGs 1d after I/R or PenCON+I/R and this is prevented by PenX5 injection in mice with I/R (n=3 per group). B. Paw guarding is increased 1d after I/R and PenCON+I/R compared to sham controls, and this is partially prevented in PenX5+I/R animals. C. Mechanical withdrawal thresholds to von Frey hair (VFH) stimulation are decreased after I/R and PenCON+I/R and this is partially recued in the PenX5+I/R group. D. Mechanical withdrawal thresholds to muscle squeezing are decreased after I/R, PenCON+I/R and PenX5+I/R. E. PenX5 injection in mice with I/R also partially inhibited I/R-related grip strength deficits observed in I/R alone or PenCON+I/R groups. F. A significant increase in mean arterial pressure (MAP) after exercise was observed in injured mice (I/R and PenCON+I/R), but this was not observed in the PenX5+I/R group. (D-E n=12 per group. x p

    Techniques Used: Injection, Mouse Assay

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    Alomone Labs p2x5 rabbit anti p2x5
    A role for peripheral GDNF signaling in the development of ischemic myalgia. After I/R, injured muscles release a variety of metabolites that include ATP, lactate and protons that stimulate group III and IV muscle afferents. Simultaneously, GDNF is being release in the injured tissues and this induces a signaling cascade in primary sensory neurons through its receptor GFRα1 that involves increased function of CREB/ CREB binding protein (CBP), which in turn modulates the observed overexpression of ATP sensitive channel <t>P2X5</t> and to a lesser degree, ASIC3. This molecular change at the DRG level in turn modulates the appearance of pain-related behaviors such as paw guarding, mechanical hypersensitivity and muscle weakness. In addition, this pathway also affects the exercise pressor reflex after injury. Targeting the overexpression of P2X5 or GFRα1 in sensory neurons or the increased GDNF levels within the muscle appears to inhibit many of these ischemic myalgia-like phenomena.
    P2x5 Rabbit Anti P2x5, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2x5 rabbit anti p2x5/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2x5 rabbit anti p2x5 - by Bioz Stars, 2022-07
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    A role for peripheral GDNF signaling in the development of ischemic myalgia. After I/R, injured muscles release a variety of metabolites that include ATP, lactate and protons that stimulate group III and IV muscle afferents. Simultaneously, GDNF is being release in the injured tissues and this induces a signaling cascade in primary sensory neurons through its receptor GFRα1 that involves increased function of CREB/ CREB binding protein (CBP), which in turn modulates the observed overexpression of ATP sensitive channel P2X5 and to a lesser degree, ASIC3. This molecular change at the DRG level in turn modulates the appearance of pain-related behaviors such as paw guarding, mechanical hypersensitivity and muscle weakness. In addition, this pathway also affects the exercise pressor reflex after injury. Targeting the overexpression of P2X5 or GFRα1 in sensory neurons or the increased GDNF levels within the muscle appears to inhibit many of these ischemic myalgia-like phenomena.

    Journal: bioRxiv

    Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

    doi: 10.1101/627521

    Figure Lengend Snippet: A role for peripheral GDNF signaling in the development of ischemic myalgia. After I/R, injured muscles release a variety of metabolites that include ATP, lactate and protons that stimulate group III and IV muscle afferents. Simultaneously, GDNF is being release in the injured tissues and this induces a signaling cascade in primary sensory neurons through its receptor GFRα1 that involves increased function of CREB/ CREB binding protein (CBP), which in turn modulates the observed overexpression of ATP sensitive channel P2X5 and to a lesser degree, ASIC3. This molecular change at the DRG level in turn modulates the appearance of pain-related behaviors such as paw guarding, mechanical hypersensitivity and muscle weakness. In addition, this pathway also affects the exercise pressor reflex after injury. Targeting the overexpression of P2X5 or GFRα1 in sensory neurons or the increased GDNF levels within the muscle appears to inhibit many of these ischemic myalgia-like phenomena.

    Article Snippet: Antibodies used for western blot were as follows for each protein: GAPDH: chicken anti-GAPDH, 1:2000 (Pro-Sci, Atlanta, GA, USA); GFRα1: goat anti-GFRα1 1:250 (cat. No. AF560, R & D Systems, Minneapolis, MN, USA); P2X5: rabbit anti-P2X5 1:800 (cat. No. APR005, Alomone Labs, Jerusalem, Israel); GDNF: rabbit anti-GDNF 1:500 (cat No. ANT-014, Alomone Labs, Jerusalem, Israel); CREB: rabbit anti-CREB 1:1000 (cat. No. ab32515, Abcam, Cambridge, MA, USA); pCREB: rabbit anti-pCREB 1:2500 (cat. No. ab32096, Abcam, Cambridge, MA, USA); CBP: rabbit anti-CBP 1:500 (cat. No. ab2832, Abcam, Cambridge, MA, USA).

    Techniques: Binding Assay, Over Expression

    Disruption of the CBP/CREB transcription factor complex regulates I/R-related pain-like behaviors, EPRs and P2X5 expression. A . pCREB and CBP are increased after I/R compared to naïve as assessed with western blotting. Penα1 injection however partially blocked the I/R-induced upregulation of pCREB and fully inhibited the upregulation of CBP in the DRGs. B. Paw guarding is increased 1d after I/R in vehicle treated (0.1% DMSO) animals and this is completely prevented by treatment with the CREB-CBP interaction antagonist xx-650-23. Mechanical withdrawal thresholds to VFH stimulation (C) and muscle squeezing (D) are significantly decreased after I/R+0.1%DMSO and I/R+xx-650-23. However, both of these I/R-induced behavioral effects were significantly blunted in xx-650-23 treated animals. E . Decreased grip strength observed 1d after I/R+0.1%DMSO was partially rescued by xx-650-23. F. Exercise pressor reflexes (reflected by changes in MAP after forced running) exacerbated by I/R were also blocked by disruption of the CBP/CREB complex G. xx-650-23 significantly reduced the levels of P2X5 in the DRGs of mice with I/R compared to I/R-injured animals treated with vehicle (0.1% DMSO). (A: n=4-7 per group, *p

    Journal: bioRxiv

    Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

    doi: 10.1101/627521

    Figure Lengend Snippet: Disruption of the CBP/CREB transcription factor complex regulates I/R-related pain-like behaviors, EPRs and P2X5 expression. A . pCREB and CBP are increased after I/R compared to naïve as assessed with western blotting. Penα1 injection however partially blocked the I/R-induced upregulation of pCREB and fully inhibited the upregulation of CBP in the DRGs. B. Paw guarding is increased 1d after I/R in vehicle treated (0.1% DMSO) animals and this is completely prevented by treatment with the CREB-CBP interaction antagonist xx-650-23. Mechanical withdrawal thresholds to VFH stimulation (C) and muscle squeezing (D) are significantly decreased after I/R+0.1%DMSO and I/R+xx-650-23. However, both of these I/R-induced behavioral effects were significantly blunted in xx-650-23 treated animals. E . Decreased grip strength observed 1d after I/R+0.1%DMSO was partially rescued by xx-650-23. F. Exercise pressor reflexes (reflected by changes in MAP after forced running) exacerbated by I/R were also blocked by disruption of the CBP/CREB complex G. xx-650-23 significantly reduced the levels of P2X5 in the DRGs of mice with I/R compared to I/R-injured animals treated with vehicle (0.1% DMSO). (A: n=4-7 per group, *p

    Article Snippet: Antibodies used for western blot were as follows for each protein: GAPDH: chicken anti-GAPDH, 1:2000 (Pro-Sci, Atlanta, GA, USA); GFRα1: goat anti-GFRα1 1:250 (cat. No. AF560, R & D Systems, Minneapolis, MN, USA); P2X5: rabbit anti-P2X5 1:800 (cat. No. APR005, Alomone Labs, Jerusalem, Israel); GDNF: rabbit anti-GDNF 1:500 (cat No. ANT-014, Alomone Labs, Jerusalem, Israel); CREB: rabbit anti-CREB 1:1000 (cat. No. ab32515, Abcam, Cambridge, MA, USA); pCREB: rabbit anti-pCREB 1:2500 (cat. No. ab32096, Abcam, Cambridge, MA, USA); CBP: rabbit anti-CBP 1:500 (cat. No. ab2832, Abcam, Cambridge, MA, USA).

    Techniques: Expressing, Western Blot, Injection, Mouse Assay

    I/R causes and increase in the total number of cells that are positive for GFRα1 and P2X5. A . Representative images of DRG sections immunostained for GFRα1 and P2X5. Yellow arrows mark GFRα1 negative cells and white arrows mark positive cells. B-D . After I/R there was a significant increase in the total number of cells immunopositive for both GFRα1 and P2X5 (B) as well as total cells positive for either GFRα1 (C) or P2X5 (D) . The increase in immunopositive cells was prevented in Penα1+I/R. (n=3 per group, *p

    Journal: bioRxiv

    Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

    doi: 10.1101/627521

    Figure Lengend Snippet: I/R causes and increase in the total number of cells that are positive for GFRα1 and P2X5. A . Representative images of DRG sections immunostained for GFRα1 and P2X5. Yellow arrows mark GFRα1 negative cells and white arrows mark positive cells. B-D . After I/R there was a significant increase in the total number of cells immunopositive for both GFRα1 and P2X5 (B) as well as total cells positive for either GFRα1 (C) or P2X5 (D) . The increase in immunopositive cells was prevented in Penα1+I/R. (n=3 per group, *p

    Article Snippet: Antibodies used for western blot were as follows for each protein: GAPDH: chicken anti-GAPDH, 1:2000 (Pro-Sci, Atlanta, GA, USA); GFRα1: goat anti-GFRα1 1:250 (cat. No. AF560, R & D Systems, Minneapolis, MN, USA); P2X5: rabbit anti-P2X5 1:800 (cat. No. APR005, Alomone Labs, Jerusalem, Israel); GDNF: rabbit anti-GDNF 1:500 (cat No. ANT-014, Alomone Labs, Jerusalem, Israel); CREB: rabbit anti-CREB 1:1000 (cat. No. ab32515, Abcam, Cambridge, MA, USA); pCREB: rabbit anti-pCREB 1:2500 (cat. No. ab32096, Abcam, Cambridge, MA, USA); CBP: rabbit anti-CBP 1:500 (cat. No. ab2832, Abcam, Cambridge, MA, USA).

    Techniques:

    Selective knockdown of P2X5 in primary muscle afferents prevents the development of pain-related behaviors and exacerbated EPRs after I/R. A . P2X5 is increased in DRGs 1d after I/R or PenCON+I/R and this is prevented by PenX5 injection in mice with I/R (n=3 per group). B. Paw guarding is increased 1d after I/R and PenCON+I/R compared to sham controls, and this is partially prevented in PenX5+I/R animals. C. Mechanical withdrawal thresholds to von Frey hair (VFH) stimulation are decreased after I/R and PenCON+I/R and this is partially recued in the PenX5+I/R group. D. Mechanical withdrawal thresholds to muscle squeezing are decreased after I/R, PenCON+I/R and PenX5+I/R. E. PenX5 injection in mice with I/R also partially inhibited I/R-related grip strength deficits observed in I/R alone or PenCON+I/R groups. F. A significant increase in mean arterial pressure (MAP) after exercise was observed in injured mice (I/R and PenCON+I/R), but this was not observed in the PenX5+I/R group. (D-E n=12 per group. x p

    Journal: bioRxiv

    Article Title: A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes

    doi: 10.1101/627521

    Figure Lengend Snippet: Selective knockdown of P2X5 in primary muscle afferents prevents the development of pain-related behaviors and exacerbated EPRs after I/R. A . P2X5 is increased in DRGs 1d after I/R or PenCON+I/R and this is prevented by PenX5 injection in mice with I/R (n=3 per group). B. Paw guarding is increased 1d after I/R and PenCON+I/R compared to sham controls, and this is partially prevented in PenX5+I/R animals. C. Mechanical withdrawal thresholds to von Frey hair (VFH) stimulation are decreased after I/R and PenCON+I/R and this is partially recued in the PenX5+I/R group. D. Mechanical withdrawal thresholds to muscle squeezing are decreased after I/R, PenCON+I/R and PenX5+I/R. E. PenX5 injection in mice with I/R also partially inhibited I/R-related grip strength deficits observed in I/R alone or PenCON+I/R groups. F. A significant increase in mean arterial pressure (MAP) after exercise was observed in injured mice (I/R and PenCON+I/R), but this was not observed in the PenX5+I/R group. (D-E n=12 per group. x p

    Article Snippet: Antibodies used for western blot were as follows for each protein: GAPDH: chicken anti-GAPDH, 1:2000 (Pro-Sci, Atlanta, GA, USA); GFRα1: goat anti-GFRα1 1:250 (cat. No. AF560, R & D Systems, Minneapolis, MN, USA); P2X5: rabbit anti-P2X5 1:800 (cat. No. APR005, Alomone Labs, Jerusalem, Israel); GDNF: rabbit anti-GDNF 1:500 (cat No. ANT-014, Alomone Labs, Jerusalem, Israel); CREB: rabbit anti-CREB 1:1000 (cat. No. ab32515, Abcam, Cambridge, MA, USA); pCREB: rabbit anti-pCREB 1:2500 (cat. No. ab32096, Abcam, Cambridge, MA, USA); CBP: rabbit anti-CBP 1:500 (cat. No. ab2832, Abcam, Cambridge, MA, USA).

    Techniques: Injection, Mouse Assay

    P2X5 subunits interact with P2X2 subunits at the plasma membrane. A , Rescue of cell surface expression of the P2X2–K366A trafficking deficient mutant by other subunits. P2X2–K366A carrying a Flag tag in the extracellular loop was expressed alone or in combination with each of the seven wild-type P2X subunits. Membrane expression was measured using a chemiluminescent assay. Cell surface expression of P2X2–K366A is rescued upon coexpression with P2X2, P2X3, P2X4, and P2X5 subunits. B , Membrane detection of the P2X5–K372A mutant carrying an extracellular Flag tag is increased when coexpressed with P2X1, P2X2, and P2X4 and P2X5 subunits. Note that because the surface expression of P2X5–K372 mutant is very low compared withP2X2–K366A, the changes appear much higher. Results are shown as mean ± SEM of at least three independent experiments. ** p

    Journal: The Journal of Neuroscience

    Article Title: P2X2 and P2X5 Subunits Define a New Heteromeric Receptor with P2X7-Like Properties

    doi: 10.1523/JNEUROSCI.6332-11.2012

    Figure Lengend Snippet: P2X5 subunits interact with P2X2 subunits at the plasma membrane. A , Rescue of cell surface expression of the P2X2–K366A trafficking deficient mutant by other subunits. P2X2–K366A carrying a Flag tag in the extracellular loop was expressed alone or in combination with each of the seven wild-type P2X subunits. Membrane expression was measured using a chemiluminescent assay. Cell surface expression of P2X2–K366A is rescued upon coexpression with P2X2, P2X3, P2X4, and P2X5 subunits. B , Membrane detection of the P2X5–K372A mutant carrying an extracellular Flag tag is increased when coexpressed with P2X1, P2X2, and P2X4 and P2X5 subunits. Note that because the surface expression of P2X5–K372 mutant is very low compared withP2X2–K366A, the changes appear much higher. Results are shown as mean ± SEM of at least three independent experiments. ** p

    Article Snippet: After preclearing, solubilized membrane proteins were incubated overnight at 4°C with either rabbit anti-P2X5 [a gift from Dr. Mark Voigt ( )] or rabbit anti-P2X2 (Alomone Labs), and incubated 1 h at 4°C with 20 μl of proteinA-Sepharose beads.

    Techniques: Expressing, Mutagenesis, FLAG-tag

    Stoichiometry of P2X2 and P2X5 interactions as assessed by BiFC and BRET/BiFC. A , Bimolecular fluorescence complementation between P2X subunits. P2X subunits fused to either the amino or carboxyl half of YFP (YN or YC) were transfected alone or in combination in HEK cells. Recomplemented fluorescence was observed by microscopy. BiFC was observed between homomeric P2X2, P2X4, and P2X5 subunits, as well as between heteromeric P2X2 and P2X5 subunits, but not between P2X2 and P2X4 subunits. Scale bar, 10 μm. B , Combination of BRET and BiFC reveals the stoichiometry of the heteromeric P2X2/P2X5 assembly. Top, Diagram illustrating the approach. BRET is only observed between one P2X2-Luc subunit and any other combinations of two P2X-hemi-YFP subunits. BRET titration curves between P2X2-Luc (left) or P2X5-Luc (right) cotransfected with P2X2, P2X5, RAMP1, and CRL fused to hemi-YFP. Specific BRET signals were observed between P2X2 and P2X5 subunits for each experimental condition, demonstrating the existence of heteromeric receptors with two different stoichiometries. Data are expressed as mean ± SEM of at least N = 3 experiments.

    Journal: The Journal of Neuroscience

    Article Title: P2X2 and P2X5 Subunits Define a New Heteromeric Receptor with P2X7-Like Properties

    doi: 10.1523/JNEUROSCI.6332-11.2012

    Figure Lengend Snippet: Stoichiometry of P2X2 and P2X5 interactions as assessed by BiFC and BRET/BiFC. A , Bimolecular fluorescence complementation between P2X subunits. P2X subunits fused to either the amino or carboxyl half of YFP (YN or YC) were transfected alone or in combination in HEK cells. Recomplemented fluorescence was observed by microscopy. BiFC was observed between homomeric P2X2, P2X4, and P2X5 subunits, as well as between heteromeric P2X2 and P2X5 subunits, but not between P2X2 and P2X4 subunits. Scale bar, 10 μm. B , Combination of BRET and BiFC reveals the stoichiometry of the heteromeric P2X2/P2X5 assembly. Top, Diagram illustrating the approach. BRET is only observed between one P2X2-Luc subunit and any other combinations of two P2X-hemi-YFP subunits. BRET titration curves between P2X2-Luc (left) or P2X5-Luc (right) cotransfected with P2X2, P2X5, RAMP1, and CRL fused to hemi-YFP. Specific BRET signals were observed between P2X2 and P2X5 subunits for each experimental condition, demonstrating the existence of heteromeric receptors with two different stoichiometries. Data are expressed as mean ± SEM of at least N = 3 experiments.

    Article Snippet: After preclearing, solubilized membrane proteins were incubated overnight at 4°C with either rabbit anti-P2X5 [a gift from Dr. Mark Voigt ( )] or rabbit anti-P2X2 (Alomone Labs), and incubated 1 h at 4°C with 20 μl of proteinA-Sepharose beads.

    Techniques: Bimolecular Fluorescence Complementation Assay, Bioluminescence Resonance Energy Transfer, Fluorescence, Transfection, Microscopy, Titration

    P2X5 and P2X2 subunits interact in neuronal tissues. A , Physical interactions of P2X2 and P2X5 subunits in brain tissues. P2X2 or P2X5 subunits were immunoprecipitated from mouse brain membrane and different subunits detected with specific antibodies. Left panel represents immunoprecipitation from total brain membrane proteins, right panel from brainstem. Note that P2X5 is not detectable from the total protein extract. Blots are representative of three independent experiments. Asterisks indicate the antibody heavy chains. B , Coimmunolocalization of P2X2 and P2X5 subunits in peripheral and central neurons. Representative images of immunohistochemistry performed on slices from DRG, spinal cord, or mid pons. P2X2 and P2X5 are stained in red and green, respectively. Note that in each structure only a subset of neurons coexpresses both subunits. Scale bar, 40 μm.

    Journal: The Journal of Neuroscience

    Article Title: P2X2 and P2X5 Subunits Define a New Heteromeric Receptor with P2X7-Like Properties

    doi: 10.1523/JNEUROSCI.6332-11.2012

    Figure Lengend Snippet: P2X5 and P2X2 subunits interact in neuronal tissues. A , Physical interactions of P2X2 and P2X5 subunits in brain tissues. P2X2 or P2X5 subunits were immunoprecipitated from mouse brain membrane and different subunits detected with specific antibodies. Left panel represents immunoprecipitation from total brain membrane proteins, right panel from brainstem. Note that P2X5 is not detectable from the total protein extract. Blots are representative of three independent experiments. Asterisks indicate the antibody heavy chains. B , Coimmunolocalization of P2X2 and P2X5 subunits in peripheral and central neurons. Representative images of immunohistochemistry performed on slices from DRG, spinal cord, or mid pons. P2X2 and P2X5 are stained in red and green, respectively. Note that in each structure only a subset of neurons coexpresses both subunits. Scale bar, 40 μm.

    Article Snippet: After preclearing, solubilized membrane proteins were incubated overnight at 4°C with either rabbit anti-P2X5 [a gift from Dr. Mark Voigt ( )] or rabbit anti-P2X2 (Alomone Labs), and incubated 1 h at 4°C with 20 μl of proteinA-Sepharose beads.

    Techniques: Immunoprecipitation, Immunohistochemistry, Staining