anti p2x4 antibodies (Alomone Labs)
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anti p2x4 antibodies
Anti P2x4 Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti p2x4 antibodies/product/Alomone Labs
Average 95 stars, based on 24 article reviews
Price from $9.99 to $1999.99
Anti P2x4 Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti p2x4 antibodies/product/Alomone Labs
Average 95 stars, based on 24 article reviews
Price from $9.99 to $1999.99
anti p2x4 antibodies - by Bioz Stars,
2022-11
95/100 stars
Images
1) Product Images from "Increased surface P2X4 receptors by mutant SOD1 proteins contribute to ALS pathogenesis in SOD1-G93A mice"
Article Title: Increased surface P2X4 receptors by mutant SOD1 proteins contribute to ALS pathogenesis in SOD1-G93A mice
Journal: Cellular and Molecular Life Sciences
doi: 10.1007/s00018-022-04461-5
Figure Legend Snippet: Surface P2X4 density is higher in macrophages of SOD1 as compared to WT mice before the onset and during the progression of the disease. A Western blotting of total and biotinylated surface proteins from peritoneal macrophages isolated from WT:WT (WT) and SOD1-G93A:WT (SOD1) mice at three time points (P40, P75 and P100). The anti-SOD1 antibody revealed 2 bands of different size corresponding to murine (m)SOD1 and human (h)SOD1-G93A confirming the genotype of the mouse. B Surface/total ratio shows that the number of surface P2X4 is increased in SOD1 macrophages as compared to WT at presymptomatic (P75) and symptomatic phase (P100). C Similar experiments from peritoneal macrophages isolated from WT:P2X4KI (P2X4KI) and SOD1:P2X4KI (SOD1KI) mice at the same 3 stages (P40, P75 and P100). D Surface/total ratio shows that the density of surface P2X4KI is similar between P2X4KI and SOD1:P2X4KI macrophages. (* p
Techniques Used: Mouse Assay, Western Blot, Isolation
Figure Legend Snippet: Absence of P2X4 ameliorates ALS motor symptoms and life survival in SOD1:P2X4KO mice. A Bar chart of the time to swim to reach a platform placed at one extremity of a corridor as a function of age (in days) for SOD1:WT (black bars) and SOD1:P2X4KO (green bars) mice (A1), (* p
Techniques Used: Mouse Assay
Figure Legend Snippet: Proposed mechanism of surface P2X4 upregulation and consequences in ALS . In normal conditions P2X4 is constitutively endocytosed by the binding of AP2 on its C-terminus internalization domain resulting in a low surface expression restricted to MN within the spinal cord. During ALS progression, (1) misfolded mutant proteins like SOD1 or TDP-43 interfere with P2X4 internalization by competing for AP2 interaction leading to an increase in surface P2X4 density in cells expressing P2X4 such as MNs and macrophages at early stages. (2) At symptomatic stages de novo P2X4 expression in spinal reactive microglia further increase P2X4 signaling in microglia. Cell-specific and time-dependent activation of P2X4 is critical for beneficial or detrimental effects on ALS
Techniques Used: Binding Assay, Expressing, Mutagenesis, Activation Assay
Figure Legend Snippet: Mutant SOD1 proteins increase surface P2X4 number and function in vitro. A Representative currents evoked by 100 µM ATP in Xenopus oocytes co-injected with cDNAs encoding the murin (m)P2X4 and either the wild-type (WT) human SOD1 (hSOD1WT) or a mutant hSOD1 (hSOD1G93A, G85R or G37R). B Mean amplitudes of ATP induced-currents computed for all tested oocytes. ATP evoked P2X4 currents are strongly increased in cells expressing mutant hSOD1 (G93A, G85R and G37R) compared to those expressing hSOD1WT (** p
Techniques Used: Mutagenesis, In Vitro, Injection, Expressing
Figure Legend Snippet: Mutant SOD1 proteins alter AP2 dependent endocytosis of P2X4 over ALS progression in the SOD1 mouse model. A Western blot analysis using anti-SOD1 antibodies after immunoprecipitation (IP) using anti-AP2 antibodies from spinal cord protein extracts of wild type (WT) and SOD1 mice at different stages (P40 to P120) revealed that SOD1-G93A co-immunoprecipitated with adaptor protein 2 (AP2) (see also panel E and Fig. S1B-C). Anti-SOD1 antibodies revealed in total proteins (input) one (mSOD1) or two bands (mSOD1 + hSOD1G93A) confirming the genotype of the mice tested. B The increase in SOD1 signals after IP over time suggests that the interaction between SOD1-G93A and AP2 increases during ALS pathogenesis (significantly different from P40, * p
Techniques Used: Mutagenesis, Western Blot, Immunoprecipitation, Mouse Assay
Figure Legend Snippet: P2X4 expression switches over the time from motoneurons to microglia within the spinal cord of SOD1 mice. A – C P2X4 immunoreactivity in lumbar spinal cords from WT, SOD1:WT (SOD1) WT:P2X4KI (P2X4KI) and SOD1:P2X4KI mice at pre- (P75) and symptomatic (P100) phases of ALS. Expression of P2X4 in WT or SOD1 mice revealed with a rat monoclonal P2X4 antibodies (Nodu-246) and expression of P2X4KI revealed with anti-RFP in P2X4KI or SOD1:P2X4KI mice at P75 and P100 (see Fig. S6). Nodu-246, Beno-271 or anti-RFP were revealed with secondary antibodies coupled to Alexa-564 (red). Neurons A, microglia B or astrocytes C were identified using primary antibodies against NeuN, GFAP or Iba1, respectively, revealed with secondary antibodies coupled to Alexa-488 (green). White frames indicate magnified areas. A P2X4 or P2X4KI are expressed in spinal neurons located in the ventral horn at P75 in SOD1 or SOD1:P2X4KI mice and at P100 solely in WT mice. B , C At P100 in SOD1 or SOD1:P2X4KI mice, the increase in Iba1 ( B) and GFAP ( C ) staining reveals the astro- and micro-gliosis within the spinal cord during ALS progression. At P100, P2X4 staining is localized in Iba1-positive cells of SOD1 or SOD1:P2X4KI spinal cords indicating the increase in P2X4 expression mainly in microglia during the symptomatic phase (See also Fig. S4 and S5)
Techniques Used: Expressing, Mouse Assay, Staining
Figure Legend Snippet: Increased surface P2X4 ameliorates ALS motor symptoms signs and life span in SOD1 internalization-defective P2X4KI mice. A Bar chart of the time to swim to a platform as a function of age (in days) for SOD1:WT (SOD1, black bars) and SOD1:P2X4KI (orange bars) mice (A1), (* p
Techniques Used: Mouse Assay
2) Product Images from "Increased surface P2X4 receptors by mutant SOD1 proteins contribute to ALS pathogenesis in SOD1-G93A mice"
Article Title: Increased surface P2X4 receptors by mutant SOD1 proteins contribute to ALS pathogenesis in SOD1-G93A mice
Journal: Cellular and Molecular Life Sciences
doi: 10.1007/s00018-022-04461-5
Figure Legend Snippet: Surface P2X4 density is higher in macrophages of SOD1 as compared to WT mice before the onset and during the progression of the disease. A Western blotting of total and biotinylated surface proteins from peritoneal macrophages isolated from WT:WT (WT) and SOD1-G93A:WT (SOD1) mice at three time points (P40, P75 and P100). The anti-SOD1 antibody revealed 2 bands of different size corresponding to murine (m)SOD1 and human (h)SOD1-G93A confirming the genotype of the mouse. B Surface/total ratio shows that the number of surface P2X4 is increased in SOD1 macrophages as compared to WT at presymptomatic (P75) and symptomatic phase (P100). C Similar experiments from peritoneal macrophages isolated from WT:P2X4KI (P2X4KI) and SOD1:P2X4KI (SOD1KI) mice at the same 3 stages (P40, P75 and P100). D Surface/total ratio shows that the density of surface P2X4KI is similar between P2X4KI and SOD1:P2X4KI macrophages. (* p
Techniques Used: Mouse Assay, Western Blot, Isolation
Figure Legend Snippet: Absence of P2X4 ameliorates ALS motor symptoms and life survival in SOD1:P2X4KO mice. A Bar chart of the time to swim to reach a platform placed at one extremity of a corridor as a function of age (in days) for SOD1:WT (black bars) and SOD1:P2X4KO (green bars) mice (A1), (* p
Techniques Used: Mouse Assay
Figure Legend Snippet: Proposed mechanism of surface P2X4 upregulation and consequences in ALS . In normal conditions P2X4 is constitutively endocytosed by the binding of AP2 on its C-terminus internalization domain resulting in a low surface expression restricted to MN within the spinal cord. During ALS progression, (1) misfolded mutant proteins like SOD1 or TDP-43 interfere with P2X4 internalization by competing for AP2 interaction leading to an increase in surface P2X4 density in cells expressing P2X4 such as MNs and macrophages at early stages. (2) At symptomatic stages de novo P2X4 expression in spinal reactive microglia further increase P2X4 signaling in microglia. Cell-specific and time-dependent activation of P2X4 is critical for beneficial or detrimental effects on ALS
Techniques Used: Binding Assay, Expressing, Mutagenesis, Activation Assay
Figure Legend Snippet: Mutant SOD1 proteins increase surface P2X4 number and function in vitro. A Representative currents evoked by 100 µM ATP in Xenopus oocytes co-injected with cDNAs encoding the murin (m)P2X4 and either the wild-type (WT) human SOD1 (hSOD1WT) or a mutant hSOD1 (hSOD1G93A, G85R or G37R). B Mean amplitudes of ATP induced-currents computed for all tested oocytes. ATP evoked P2X4 currents are strongly increased in cells expressing mutant hSOD1 (G93A, G85R and G37R) compared to those expressing hSOD1WT (** p
Techniques Used: Mutagenesis, In Vitro, Injection, Expressing
Figure Legend Snippet: Mutant SOD1 proteins alter AP2 dependent endocytosis of P2X4 over ALS progression in the SOD1 mouse model. A Western blot analysis using anti-SOD1 antibodies after immunoprecipitation (IP) using anti-AP2 antibodies from spinal cord protein extracts of wild type (WT) and SOD1 mice at different stages (P40 to P120) revealed that SOD1-G93A co-immunoprecipitated with adaptor protein 2 (AP2) (see also panel E and Fig. S1B-C). Anti-SOD1 antibodies revealed in total proteins (input) one (mSOD1) or two bands (mSOD1 + hSOD1G93A) confirming the genotype of the mice tested. B The increase in SOD1 signals after IP over time suggests that the interaction between SOD1-G93A and AP2 increases during ALS pathogenesis (significantly different from P40, * p
Techniques Used: Mutagenesis, Western Blot, Immunoprecipitation, Mouse Assay
Figure Legend Snippet: P2X4 expression switches over the time from motoneurons to microglia within the spinal cord of SOD1 mice. A – C P2X4 immunoreactivity in lumbar spinal cords from WT, SOD1:WT (SOD1) WT:P2X4KI (P2X4KI) and SOD1:P2X4KI mice at pre- (P75) and symptomatic (P100) phases of ALS. Expression of P2X4 in WT or SOD1 mice revealed with a rat monoclonal P2X4 antibodies (Nodu-246) and expression of P2X4KI revealed with anti-RFP in P2X4KI or SOD1:P2X4KI mice at P75 and P100 (see Fig. S6). Nodu-246, Beno-271 or anti-RFP were revealed with secondary antibodies coupled to Alexa-564 (red). Neurons A, microglia B or astrocytes C were identified using primary antibodies against NeuN, GFAP or Iba1, respectively, revealed with secondary antibodies coupled to Alexa-488 (green). White frames indicate magnified areas. A P2X4 or P2X4KI are expressed in spinal neurons located in the ventral horn at P75 in SOD1 or SOD1:P2X4KI mice and at P100 solely in WT mice. B , C At P100 in SOD1 or SOD1:P2X4KI mice, the increase in Iba1 ( B) and GFAP ( C ) staining reveals the astro- and micro-gliosis within the spinal cord during ALS progression. At P100, P2X4 staining is localized in Iba1-positive cells of SOD1 or SOD1:P2X4KI spinal cords indicating the increase in P2X4 expression mainly in microglia during the symptomatic phase (See also Fig. S4 and S5)
Techniques Used: Expressing, Mouse Assay, Staining
Figure Legend Snippet: Increased surface P2X4 ameliorates ALS motor symptoms signs and life span in SOD1 internalization-defective P2X4KI mice. A Bar chart of the time to swim to a platform as a function of age (in days) for SOD1:WT (SOD1, black bars) and SOD1:P2X4KI (orange bars) mice (A1), (* p
Techniques Used: Mouse Assay
3) Product Images from "Persistent elevation of lysophosphatidylcholine promotes radiation brain necrosis with microglial recruitment by P2RX4 activation"
Article Title: Persistent elevation of lysophosphatidylcholine promotes radiation brain necrosis with microglial recruitment by P2RX4 activation
Journal: Scientific Reports
doi: 10.1038/s41598-022-12293-3
Figure Legend Snippet: Immunostaining of Iba-1 and P2RX4 between non-treated and Ivermectin (IVE)-treated RN model mice. Histology of the non-irradiated (C) and irradiated (R) areas using ( A ) Iba-1 antibodies and ( B ) P2RX4 antibodies in the non-treated and IVE-treated brain radiation necrosis mice at 5.5 months after irradiation. Bar graphs indicate the numbers in each group (non-treated: n = 6 and IVE-treated: n = 7). The same is shown for mice at 9 months after irradiation (non-treated: n = 6 and IVE-treated: n = 6). Scale bar = 50 µm. *** P
Techniques Used: Immunostaining, Mouse Assay, Irradiation
4) Product Images from "Microglial P2X4 receptors promote ApoE degradation and cognitive deficits in Alzheimer disease"
Article Title: Microglial P2X4 receptors promote ApoE degradation and cognitive deficits in Alzheimer disease
Journal: bioRxiv
doi: 10.1101/2022.05.12.491601
Figure Legend Snippet: Characterization of the interaction between P2X4 and ApoE in recombinant system. (A) Representative immunofluorescence of ApoE (green) and P2X4 (red), and DAPI (blue) in co-transfected COS-7 cells. Both ApoE and P2X4 co-localize in intracellular compartments. Scale bar 10 μm. (B, C) Comparison of ApoE expression upon co-transfection with P2X4. COS-7 cells were transfected with ApoE alone or in combination with P2X4. Expression of ApoE was analyzed by Western blot in both cell culture supernatants and cell lysates (B). Quantitative analysis shows that in the presence of P2X4, amounts of ApoE is reduced in both culture supernatant (Sup) and cell lysates (Lys) (C). n = 3 independent experiments, ** p
Techniques Used: Recombinant, Immunofluorescence, Transfection, Expressing, Cotransfection, Western Blot, Cell Culture
Figure Legend Snippet: Increased ApoE in microglia from APP/PS1xKO mice and AD patients. (A) Immunofluorescence of ApoE (blue), Iba1 (green) and P2X4 (red) in APP/PS1 mice cortex. Scale bar 10 μm. (B) Immunofluorescence of CD68 (red), Iba1 (green), P2X4 (white) in APP/PS1 mice cortex. Scale bar 20 μm. (C, D, E) Analysis of ApoE expression in FACS sorted microglia from APP/PS1 and APP/PS1xKO mice. (C) Microglia were sorted based on CD11b-PE positive selection. (D) Representative western blot of ApoE from APP/PS1 and APP/PS1xKO FACS-sorted cortical microglia. (E) Quantitative analysis of signals presented in C shows an increase in ApoE in APP/PS1xKO mice relative to APP/PS1 mice. N = 2 independent experiments, n = 2 mice per group. (F) Representative pictures of cortical brain slices from healthy donor and AD patients labeled with AmyloGlo (blue, amyloid plaques), ApoE (green) and Iba1 (red) showing an increased expression of ApoE in human microglia clustered around amyloid deposit. Scale bar 20 μm.
Techniques Used: Mouse Assay, Immunofluorescence, Expressing, FACS, Selection, Western Blot, Labeling
Figure Legend Snippet: P2X4 is specifically expressed in plaque associated microglia in both human and mice AD brain. (A) Representative pictures of cortical brain slices from AD patients and healthy control labeled with AmyloGlo (blue, amyloid plaques), P2X4 (green) and Iba1 (red). P2X4 staining co-localizes with Iba1 in regions of dense amyloid plaque staining, supporting that microglia clustered around amyloid deposit specifically express P2X. In healthy control brain, P2X4 staining does not co-localizes with that of Iba1. Scale bar 20 μm. (B) Representative low magnification picture of immunofluorescence showing P2X4 (red) and Iba1 (green) immunostaining in the cortex of 12 months APP/PS1 mice. Both P2X4 and Iba1 staining co-localize in spot corresponding to amyloid plaques. Scale bar 200 μm. (C) High magnification of P2X4 (red) Iba1 (green) immunostaining at the vicinity of amyloid plaques (Amylo Glo staining, blue) in the cortex of 12 APP/PS1 mice (top) and APP/PS1xKO mice ( bottom ). Note the specific intracellular localization of P2X4 in microglia clustered around amyloid deposit. Scale bar 20 μm. (D) Representative immunofluorescence in APP/PS1 mice showing that parenchymal microglia (Iba1, green) do not express P2X4 (red) in region with no amyloid deposit (Amylo Glo staining, blue). Scale bar 20 μm.
Techniques Used: Mouse Assay, Labeling, Staining, Immunofluorescence, Immunostaining
Figure Legend Snippet: P2X4 interacts with ApoE in BMDM endo-lysosomal compartments and reduces its amount compared to P2X4-deficient cells. ( A, B ). Co-immunoprecipitation of P2X4 and ApoE. BMDM membrane extracts from WT and KO mice were immunoprecipitated (IP) with an anti-P2X4 antibody (A), or ApoE antibody (B). Immunoprecipitated proteins were separated by electrophoresis and immunoblotted with either anti-ApoE (top row) or anti-P2X4 (bottom row) antibodies. (C) Representative immunofluorescence image showing the co-localization of the lysosomal marker CD68 (green), P2X4 (red) and ApoE (purple) in BMDM cells. Scale bar 5 μm. (D, E) Western blot analysis of ApoE in BMDM culture supernatants (Sup) or cell lysates (Lys) from WT and KO mice. (D) Representative western blot of ApoE, (E) Quantification of western blot presented in D. A significant increase of ApoE is observed in both KO cultures supernatants and in cell lysates. Results were normalized to ApoE signal obtained for WT BMDM in each culture. n = 6 independent cultures, * p
Techniques Used: Immunoprecipitation, Mouse Assay, Electrophoresis, Immunofluorescence, Marker, Western Blot
Figure Legend Snippet: P2X4 regulates cathepsin B-dependent ApoE degradation. (A, B) Comparison of treatment with E64 a pharmacological inhibitor of the cysteine proteases, on ApoE expression in BMDM culture of WT and P2X4 -/- mice. (A) Representative western blot of ApoE in the supernatant of WT and P2X4 -/- BMDM after incubation with 10 μM E64. (B) Quantitative analysis of western blots shows that E64 induced a strong increase of ApoE in the supernatant of WT but not in P2X4 -/- BMDM. n= 5 independent experiments, ** p
Techniques Used: Expressing, Mouse Assay, Western Blot, Incubation
Figure Legend Snippet: Deletion of p2x4 does not affect amyloid plaques density but reduces soluble Aß species. (A) Representative images of Thioflavine T staining in APP/PS1 and APP/PS1xKO brain. Scale bar 700 μm. (B) Cumulative frequency of the range size of amyloid plaques. There is no obvious difference in the number of plaques not of their size between APP/PS1 and APP/PS1xKO; n = 11 mice per group. (C and D) Analysis of microglial clustering around amyloid plaque between in the cortex of APP/PS1 and APP/PS1xKO mice. (C) Representative image of microglia clustering around plaques. Amyloid plaques are stained in blue and Iba1 is in green. Scale bar 20 μm. (D) Quantification of the area covered by microglia surrounding amyloid plaques. The ratio of the surface occupied by microglia over the surface of the plaque is expressed for both APP/PS1 and APP/PS1xKO mice. n = 11 mice per group, unpaired t-test. (E) Representative Western blot of Aß peptide detected with the 6E10 antibody from cortex extracts from APP/PS1 and APP/PS1xKO mice. (F) Quantitative analysis of Western blots presented in (E). A significant decrease of the Aβ peptide amount is observed in APP/PS1xKO mice. n = 7 mice per group, * p
Techniques Used: Staining, Mouse Assay, Western Blot
Figure Legend Snippet: p2x4 deletion reverses memory deficit in APP/PS1 mice. (A) Left , Latency to locate the drink house 15 h after water deprivation in the Hamlet test. WT and KO mice present reduced latency to the drink house, whereas no difference was observed between non-water deprived (NWD) and water-deprived (WD) conditions in APP/PS1 mice. APP/PS1xKO water deprived mice present significant reduction of the latency, indicating that mice have retained the location of the drink house. Right , Number of errors before entering the drink house. WT and KO mice present reduced number of errors, whereas no difference was observed between non-water and water-deprived condition in APP/PS1 mice. APP/PS1xKO deprived-water mice present significant reduction of the number of errors. N = 3 independent experiments, n = 8-11 mice per group. * p
Techniques Used: Mouse Assay
5) Product Images from "Microglial P2X4 receptors promote ApoE degradation and cognitive deficits in Alzheimer disease"
Article Title: Microglial P2X4 receptors promote ApoE degradation and cognitive deficits in Alzheimer disease
Journal: bioRxiv
doi: 10.1101/2022.05.12.491601
Figure Legend Snippet: Characterization of the interaction between P2X4 and ApoE in recombinant system. (A) Representative immunofluorescence of ApoE (green) and P2X4 (red), and DAPI (blue) in co-transfected COS-7 cells. Both ApoE and P2X4 co-localize in intracellular compartments. Scale bar 10 μm. (B, C) Comparison of ApoE expression upon co-transfection with P2X4. COS-7 cells were transfected with ApoE alone or in combination with P2X4. Expression of ApoE was analyzed by Western blot in both cell culture supernatants and cell lysates (B). Quantitative analysis shows that in the presence of P2X4, amounts of ApoE is reduced in both culture supernatant (Sup) and cell lysates (Lys) (C). n = 3 independent experiments, ** p
Techniques Used: Recombinant, Immunofluorescence, Transfection, Expressing, Cotransfection, Western Blot, Cell Culture
Figure Legend Snippet: Increased ApoE in microglia from APP/PS1xKO mice and AD patients. (A) Immunofluorescence of ApoE (blue), Iba1 (green) and P2X4 (red) in APP/PS1 mice cortex. Scale bar 10 μm. (B) Immunofluorescence of CD68 (red), Iba1 (green), P2X4 (white) in APP/PS1 mice cortex. Scale bar 20 μm. (C, D, E) Analysis of ApoE expression in FACS sorted microglia from APP/PS1 and APP/PS1xKO mice. (C) Microglia were sorted based on CD11b-PE positive selection. (D) Representative western blot of ApoE from APP/PS1 and APP/PS1xKO FACS-sorted cortical microglia. (E) Quantitative analysis of signals presented in C shows an increase in ApoE in APP/PS1xKO mice relative to APP/PS1 mice. N = 2 independent experiments, n = 2 mice per group. (F) Representative pictures of cortical brain slices from healthy donor and AD patients labeled with AmyloGlo (blue, amyloid plaques), ApoE (green) and Iba1 (red) showing an increased expression of ApoE in human microglia clustered around amyloid deposit. Scale bar 20 μm.
Techniques Used: Mouse Assay, Immunofluorescence, Expressing, FACS, Selection, Western Blot, Labeling
Figure Legend Snippet: P2X4 is specifically expressed in plaque associated microglia in both human and mice AD brain. (A) Representative pictures of cortical brain slices from AD patients and healthy control labeled with AmyloGlo (blue, amyloid plaques), P2X4 (green) and Iba1 (red). P2X4 staining co-localizes with Iba1 in regions of dense amyloid plaque staining, supporting that microglia clustered around amyloid deposit specifically express P2X. In healthy control brain, P2X4 staining does not co-localizes with that of Iba1. Scale bar 20 μm. (B) Representative low magnification picture of immunofluorescence showing P2X4 (red) and Iba1 (green) immunostaining in the cortex of 12 months APP/PS1 mice. Both P2X4 and Iba1 staining co-localize in spot corresponding to amyloid plaques. Scale bar 200 μm. (C) High magnification of P2X4 (red) Iba1 (green) immunostaining at the vicinity of amyloid plaques (Amylo Glo staining, blue) in the cortex of 12 APP/PS1 mice (top) and APP/PS1xKO mice ( bottom ). Note the specific intracellular localization of P2X4 in microglia clustered around amyloid deposit. Scale bar 20 μm. (D) Representative immunofluorescence in APP/PS1 mice showing that parenchymal microglia (Iba1, green) do not express P2X4 (red) in region with no amyloid deposit (Amylo Glo staining, blue). Scale bar 20 μm.
Techniques Used: Mouse Assay, Labeling, Staining, Immunofluorescence, Immunostaining
Figure Legend Snippet: P2X4 interacts with ApoE in BMDM endo-lysosomal compartments and reduces its amount compared to P2X4-deficient cells. ( A, B ). Co-immunoprecipitation of P2X4 and ApoE. BMDM membrane extracts from WT and KO mice were immunoprecipitated (IP) with an anti-P2X4 antibody (A), or ApoE antibody (B). Immunoprecipitated proteins were separated by electrophoresis and immunoblotted with either anti-ApoE (top row) or anti-P2X4 (bottom row) antibodies. (C) Representative immunofluorescence image showing the co-localization of the lysosomal marker CD68 (green), P2X4 (red) and ApoE (purple) in BMDM cells. Scale bar 5 μm. (D, E) Western blot analysis of ApoE in BMDM culture supernatants (Sup) or cell lysates (Lys) from WT and KO mice. (D) Representative western blot of ApoE, (E) Quantification of western blot presented in D. A significant increase of ApoE is observed in both KO cultures supernatants and in cell lysates. Results were normalized to ApoE signal obtained for WT BMDM in each culture. n = 6 independent cultures, * p
Techniques Used: Immunoprecipitation, Mouse Assay, Electrophoresis, Immunofluorescence, Marker, Western Blot
Figure Legend Snippet: P2X4 regulates cathepsin B-dependent ApoE degradation. (A, B) Comparison of treatment with E64 a pharmacological inhibitor of the cysteine proteases, on ApoE expression in BMDM culture of WT and P2X4 -/- mice. (A) Representative western blot of ApoE in the supernatant of WT and P2X4 -/- BMDM after incubation with 10 μM E64. (B) Quantitative analysis of western blots shows that E64 induced a strong increase of ApoE in the supernatant of WT but not in P2X4 -/- BMDM. n= 5 independent experiments, ** p
Techniques Used: Expressing, Mouse Assay, Western Blot, Incubation
Figure Legend Snippet: Deletion of p2x4 does not affect amyloid plaques density but reduces soluble Aß species. (A) Representative images of Thioflavine T staining in APP/PS1 and APP/PS1xKO brain. Scale bar 700 μm. (B) Cumulative frequency of the range size of amyloid plaques. There is no obvious difference in the number of plaques not of their size between APP/PS1 and APP/PS1xKO; n = 11 mice per group. (C and D) Analysis of microglial clustering around amyloid plaque between in the cortex of APP/PS1 and APP/PS1xKO mice. (C) Representative image of microglia clustering around plaques. Amyloid plaques are stained in blue and Iba1 is in green. Scale bar 20 μm. (D) Quantification of the area covered by microglia surrounding amyloid plaques. The ratio of the surface occupied by microglia over the surface of the plaque is expressed for both APP/PS1 and APP/PS1xKO mice. n = 11 mice per group, unpaired t-test. (E) Representative Western blot of Aß peptide detected with the 6E10 antibody from cortex extracts from APP/PS1 and APP/PS1xKO mice. (F) Quantitative analysis of Western blots presented in (E). A significant decrease of the Aβ peptide amount is observed in APP/PS1xKO mice. n = 7 mice per group, * p
Techniques Used: Staining, Mouse Assay, Western Blot
Figure Legend Snippet: p2x4 deletion reverses memory deficit in APP/PS1 mice. (A) Left , Latency to locate the drink house 15 h after water deprivation in the Hamlet test. WT and KO mice present reduced latency to the drink house, whereas no difference was observed between non-water deprived (NWD) and water-deprived (WD) conditions in APP/PS1 mice. APP/PS1xKO water deprived mice present significant reduction of the latency, indicating that mice have retained the location of the drink house. Right , Number of errors before entering the drink house. WT and KO mice present reduced number of errors, whereas no difference was observed between non-water and water-deprived condition in APP/PS1 mice. APP/PS1xKO deprived-water mice present significant reduction of the number of errors. N = 3 independent experiments, n = 8-11 mice per group. * p
Techniques Used: Mouse Assay
6) Product Images from "Synergistic Cytokine Production by ATP and PGE2 via P2X4 and EP3 Receptors in Mouse Bone-Marrow-Derived Mast Cells"
Article Title: Synergistic Cytokine Production by ATP and PGE2 via P2X4 and EP3 Receptors in Mouse Bone-Marrow-Derived Mast Cells
Journal: Cells
doi: 10.3390/cells11040616
Figure Legend Snippet: Effects of PGE 2 , ATP, and co-stimulation of PGE 2 and ATP on ERK 1/2, Akt and NF-κB phosphorylation in the BMMCs. ( A , B ) The BMMCs prepared from WT ( A ) and P2rx4 −/− mice ( B ) were stimulated with PGE 2 (0.1 μM), ATP (100 μM), and PGE 2 and ATP for 5, 10, and 20 min. Cell lysates were subjected to Western blot analysis for phospho-Akt and total Akt (upper), phospho-ERK 1/2 and total-ERK 1/2 (middle), or phospho-NF-κB and total-NF-κB (lower). Blots are representative of three independent experiments. ( C ) The densitometry value of protein bands of phospho-Akt ( C ), phospho-ERK 1/2 ( D ), and phospho-NF-κB ( E ) are shown as relative intensities, with the results obtained with unstimulated cells (Time 0) designated as 1. Data are shown as mean ± SEM ( n = 3). The effect of co-stimulation of PGE 2 and ATP on ERK 1/2, Akt, and NF-κB phosphorylation is significantly different from that of PGE 2 alone. * p
Techniques Used: Mouse Assay, Western Blot
Figure Legend Snippet: Lack of ATP-induced upregulation of PGE 2 -induced cytokine production in P2X4R-deficient ( P2rx4 −/− ) mice. The BMMCs, prepared from wild-type (WT) and P2rx4 −/− mice, were stimulated with vehicle (None) or PGE 2 (0.1 μM) in the presence or absence of ATP (100 μM) for ( A – C ) 1 h or ( D – F ) 3 h. mRNA expression for ( A ) IL-6, ( B ) IL-13, and ( C ) TNF-α were examined by quantitative RT-PCR. Data were normalized with GAPDH mRNA levels. Values are shown as mean ± SEM ( n = 3). ** p
Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR
7) Product Images from "Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms"
Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms
Journal: International Journal of Molecular Sciences
doi: 10.3390/ijms222010970
Figure Legend Snippet: Proposed mechanism for EV action on microglial cells. EVs carrying MFG-E8 proteins associated with phosphatidylserine exposed on the outer membrane are recognized by the αVβ3/αVβ5 integrin receptors of microglial cells and trigger lipid raft formation, interaction with P2X4 receptors, and possibly other molecules enriched in the lipid rafts such as components of the TLR4 multireceptor complex. These events lead to the upregulation of intracellular Ca 2+ , release of ATP, and increased motility of microglia.
Techniques Used:
Figure Legend Snippet: Co-immunoprecipitation of MFG-E8 and P2X4. Representative Western blots showing co-immunoprecipitation of MFG-E8 and P2X4R proteins in human microglial cells treated (or not) with EVs for 2 h, + indicates treatment with appropriate antibody. Full blots are available in Supplementary Figure S4 .
Techniques Used: Immunoprecipitation, Western Blot
8) Product Images from "Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms"
Article Title: Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms
Journal: International Journal of Molecular Sciences
doi: 10.3390/ijms222010970
Figure Legend Snippet: Proposed mechanism for EV action on microglial cells. EVs carrying MFG-E8 proteins associated with phosphatidylserine exposed on the outer membrane are recognized by the αVβ3/αVβ5 integrin receptors of microglial cells and trigger lipid raft formation, interaction with P2X4 receptors, and possibly other molecules enriched in the lipid rafts such as components of the TLR4 multireceptor complex. These events lead to the upregulation of intracellular Ca 2+ , release of ATP, and increased motility of microglia.
Techniques Used:
Figure Legend Snippet: Co-immunoprecipitation of MFG-E8 and P2X4. Representative Western blots showing co-immunoprecipitation of MFG-E8 and P2X4R proteins in human microglial cells treated (or not) with EVs for 2 h, + indicates treatment with appropriate antibody. Full blots are available in Supplementary Figure S4 .
Techniques Used: Immunoprecipitation, Western Blot
9) Product Images from "P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors"
Article Title: P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors
Journal: International Journal of Molecular Sciences
doi: 10.3390/ijms221910492
Figure Legend Snippet: Colocalization of P2X4 receptors with endolysosomal markers. rP2X4-EGFP receptors were expressed in NRK cells, and live cells were imaged by confocal fluorescence microscopy 48 h post transfection after co-labeling lysosomes with a variety of different markers. These include ( A ) LAMP1-mCherry (LAMP1-mCh), which was co-transfected with rP2X4-EGFP, and ( B ) Texas Red-labeled Dextran (DexTR) incubated for 5 h and chased for 2 h at 37 °C. ( C ) Calculation of Pearson’s coefficient showed the high degree of colocalization between rP2X4 and LAMP1-mCh/DexTR. Results are the mean ± SEM from three independent experiments, total number of cells, n = 40. ( D ) Cells were incubated with Lysotracker (50 nM) for 10 min at 37 °C and, in ( E , F ), with a fluorescent substrate of cathepsin B, Magic Red TM (MR) for 5 min. Cells in ( F ) were imaged using TIRF microscopy to show only those rP2X4- and MR-positive compartments located within ~100 nm of the plasma membrane. Arrowheads indicate examples of compartments that are labeled only by rP2X4 or MR. Scale bars are 10 µm (5 µm for enlargements).
Techniques Used: Fluorescence, Microscopy, Transfection, Labeling, Incubation
![Expression of P2X4 alters histamine-evoked cytosolic Ca 2+ signals measured at individual lysosomes. ( A ) Representative widefield images of HeLa cells co-expressing LAMP1-GECO with rP2X4 before and after stimulation with 50 µM histamine in Ca 2+ free HBS. Scale bar = 10 µm. ( B ) Lysosomes were tracked during the experiment to elucidate [Ca 2+ ] c around single lysosomes, and examples of the trajectories of two different lysosomes that were tracked during a single experiment are shown. ( C , D ) Typical histamine-evoked responses recorded from single tracked lysosomes are shown for cells expressing LAMP1-GECO alone or co-expressing LAMP1-GECO with rP2X4. ( E ) There was no significant difference in the baseline fluorescence. ( F ) Percentage of tracks showing a double peak following stimulation with histamine (50 µM) was greater in cells co-expressing rP2X4 with LAMP1-GECO, as was the area under the curve for the first peak ( G ). The total number of tracks was 57 (LAMP1-GECO alone) and 63 (LAMP1-GECO with rP2X4) collected over 3–6 independent experiments; unpaired Student’s t -test, * p](https://storage.googleapis.com/bioz_article_images/PMC8508626/ijms-22-10492-g004.jpg "Expression of P2X4 alters histamine-evoked cytosolic Ca 2+ signals measured at ...")
Figure Legend Snippet: Expression of P2X4 alters histamine-evoked cytosolic Ca 2+ signals measured at individual lysosomes. ( A ) Representative widefield images of HeLa cells co-expressing LAMP1-GECO with rP2X4 before and after stimulation with 50 µM histamine in Ca 2+ free HBS. Scale bar = 10 µm. ( B ) Lysosomes were tracked during the experiment to elucidate [Ca 2+ ] c around single lysosomes, and examples of the trajectories of two different lysosomes that were tracked during a single experiment are shown. ( C , D ) Typical histamine-evoked responses recorded from single tracked lysosomes are shown for cells expressing LAMP1-GECO alone or co-expressing LAMP1-GECO with rP2X4. ( E ) There was no significant difference in the baseline fluorescence. ( F ) Percentage of tracks showing a double peak following stimulation with histamine (50 µM) was greater in cells co-expressing rP2X4 with LAMP1-GECO, as was the area under the curve for the first peak ( G ). The total number of tracks was 57 (LAMP1-GECO alone) and 63 (LAMP1-GECO with rP2X4) collected over 3–6 independent experiments; unpaired Student’s t -test, * p
Techniques Used: Expressing, Fluorescence
Figure Legend Snippet: Stimulation of P2X7 receptors promotes endolysosome alkalinization and an enhanced P2X4 receptor-dependent Ca 2+ signal. ( A ) Representative images of cells co-expressing rP2X4 and rP2X7 with endolysosomes labeled with the pH-sensitive Oregon Green 488 10 kD Dextran (DexOG) and pH-insensitive DexTR either with or without treatment with 30 µM BzATP for 0.5 h. ( B ) The fluorescence ratio of DexOG to DexTR per lysosome was increased in response to BzATP addition. The mean and SEM of this ratio for control and BzATP treated cells is shown for three independent experiments (557–2050 lysosomes per experiment). ( C ) Colocalization between LAMP1-GECO- and DexTR-positive compartments. ( D ) Changes in mean cellular LAMP1-GECO fluorescence during application of 30 µM BzATP in HBS is shown for cells expressing rP2X4 and rP2X7 receptors individually or together, or rP2X7 with the rP2X4K6A mutant, as indicated. The mean cellular fluorescence ± SEM was obtained from 50 cells per condition from a single experiment, and, for each cell, the response was normalized to the maximal response (F max ) obtained by a final addition of ionomycin plus 5 mM Ca 2+ . ( E ) The mean peak amplitudes of the BzATP-evoked responses normalized to F max , from three separate experiments, are plotted for all of the receptor combinations. Results are the mean ± SEM. * p
Techniques Used: Expressing, Labeling, Fluorescence, Mutagenesis
Figure Legend Snippet: Activation of plasma membrane P2X7 receptors increases endolysosome size and trafficking in a P2X4 receptor-dependent manner. ( A ) In NRK cells expressing rP2X4-EGFP and rP2X7 receptors, endolysosomes were labeled with DexTR prior to treatment with 30 µM BzATP for 0.5 h at 37 °C. ( B ) A histogram of the percentage of cells with two or more enlarged DexTR positive lysosomes (≥1.8 µm) for control and BzATP-stimulated conditions. Stimulation with BzATP was carried out in normal DMEM (2 mM Ca 2+ ) or zero Ca 2+ DMEM. ( C ) Representative images of DexTR-labeled lysosomes in cells expressing the human isoform of P2X7 alone, in combination with rP2X4-EGFP, the rP2X4K67A-EGFP mutant, or rP2X4-EGFP alone. Control cells and those treated with 100 µM BzATP in DMEM for 0.5 h at 37 °C are shown. In cells expressing hP2X7 alone, EGFP was co-expressed to identify transfected cells. The frequency distribution of lysosome size for control and BzATP-stimulated cells is shown for cells co-expressing rP2X4 and hP2X7 receptors ( D ), expressing hP2X7 alone ( E ), and expressing rP2X4 alone ( F ). For cells expressing rP2X4 alone, a comparison was also made with cells treated with MgATP (100 µM) for 0.5 h at 37 °C. For statistical analysis, control versus agonist-stimulated was compared for each of the size groups. ( G ) To investigate lysosome trafficking, cells expressing either rP2X4 alone or co-expressing either wild-type rP2X4 or rP2X4-K67A with hP2X7 were treated with BzATP (100 µM, 0.5 h) and dynasore (80 µM, 0.5 h), fixed, and immunostained with an anti-P2X4 antibody and a FITC-labeled anti-rabbit secondary antibody. The nucleus was stained with DapI. ( H ) The distribution of rP2X4 was assessed by calculating the perinuclear index, which provides a measure of the proportion of receptors in the vicinity of the nucleus ( ≤ 5 µm) compared to those with a peripheral distribution ( ≥ 5 µm). All results are the mean ± SEM from three independent experiments. * p
Techniques Used: Activation Assay, Expressing, Labeling, Mutagenesis, Transfection, Staining
10) Product Images from "The mechanism behind activation of the Nod-like receptor family protein 3 inflammasome in Parkinson’s disease"
Article Title: The mechanism behind activation of the Nod-like receptor family protein 3 inflammasome in Parkinson’s disease
Journal: Neural Regeneration Research
doi: 10.4103/1673-5374.323077
Figure Legend Snippet: Effect of the lentivirus carrying siRNA for P2X4R on the protein expression levels of P2X4R (A), NLRP3 (B), caspase-1 (C), IL-1β (D), and IL-18 (E) in the substantia nigra pars compacta . Data are expressed as mean ± SEM ( n = 8 mice/group). ** P
Techniques Used: Expressing, Mouse Assay
![Effect of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro3,2-e]-1,4-diazepin-2-one (5-BDBD) on the protein expression of P2X4R (A), NLRP3 (B), caspase-1 (C), IL-1β (D), and IL-18 (E) in the substantia nigra pars compacta . Data are expressed as mean ± SEM ( n = 8 mice/group). ** P](https://storage.googleapis.com/bioz_article_images/PMC8530148/NRR-17-898-g004.jpg "... (5-BDBD) on the protein expression of P2X4R (A), NLRP3 (B), caspase-1 (C), IL-1β (D), and IL-18 (E) ...")
Figure Legend Snippet: Effect of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro3,2-e]-1,4-diazepin-2-one (5-BDBD) on the protein expression of P2X4R (A), NLRP3 (B), caspase-1 (C), IL-1β (D), and IL-18 (E) in the substantia nigra pars compacta . Data are expressed as mean ± SEM ( n = 8 mice/group). ** P
Techniques Used: Expressing, Mouse Assay
Figure Legend Snippet: Effect of a lentivirus carrying P2X4R on the protein expression levels of P2X4R (A), NLRP3 (B), caspase-1 (C), IL-1β (D), and IL-18 (E) in the substantia nigra pars compacta . Data are expressed as mean ± SEM ( n = 8 mice/group). ** P
Techniques Used: Expressing, Mouse Assay
11) Product Images from "P2X4 Purinergic Receptors as a Therapeutic Target in Aggressive Prostate Cancer"
Article Title: P2X4 Purinergic Receptors as a Therapeutic Target in Aggressive Prostate Cancer
Journal: bioRxiv
doi: 10.1101/2021.06.04.446195
Figure Legend Snippet: There is elevated P2X4 receptor protein expression in (A) cancer compared to benign tissues (n = 456; p
Techniques Used: Expressing
Figure Legend Snippet: P2X4 purinergic receptor protein is expressed (A) modestly in the epithelium of prostate glands and more intensely on immune cells of organ donor prostates (C) modestly in benign glands and more intensely in prostatic intraepithelial neoplasia (PIN) and cancer in prostatectomy samples and (D) on immune cells. (B) H E staining confirmed tissue types. (E) Dual staining showed that tryptase positive mast cells (red) do not express P2X4 protein (brown), but some CD66 positive neutrophils (brown) and most CD68 positive macrophages (brown) express P2X4 receptors (red). Images are at 40x magnification unless otherwise stated.
Techniques Used: Staining
Figure Legend Snippet: (A, B) P2X4 mRNA expression was detected in cancer and immune cells and in various metastatic sites. (C) P2X4 mRNA expression (brown) detected on CD68 + (red) macrophages in metastatic tissues. (D) There is a positive correlation between CD68 and P2X4 mRNA expression in metastatic tissues.
Techniques Used: Expressing
Figure Legend Snippet: (A) P2X4 antagonist 5-BDBD resulted in a dose-dependent decrease in PCa cell viability. (B) P2X4 down in LNCaP cells resulted attenuated cell growth over time. (C) Treatment with 5-BDBD resulted in a dose-dependent induction of cleaved caspase-3 in PCa cells.
Techniques Used:
Figure Legend Snippet: (A) Nucleotide treatment and (B) P2X4 knock down in DU145 cells. (C) CTP treatment increased cell migration and invasion while 5-BDBD and P2X4 knock down decreased cell migration and invasion in transwell assays. (P = parental, KD = knockdown)
Techniques Used: Migration
Figure Legend Snippet: (A) Robust P2X4 mRNA expression is detected in mouse prostate tumors. (B) CRISPR knockdown of P2X4 receptor expression in Myc-CaP cells (n=10) resulted in attenuated allograft growth in FVB/NJ mice compared to control Myc-CaP cells (n=10). (C) Human c-Myc RISH and P2X4 and AR IHC analysis confirmed P2X4 reduced expression in Myc-CaP allograft tissue from mice. (D) P2X4 positive regions lacking AR and human c-Myc stain positive for CD11b and CD3 in Myc-CaP allograft tissue from mice.
Techniques Used: Expressing, CRISPR, Mouse Assay, Immunohistochemistry, Staining
12) Product Images from "P2X4 Purinergic Receptors as a Therapeutic Target in Aggressive Prostate Cancer"
Article Title: P2X4 Purinergic Receptors as a Therapeutic Target in Aggressive Prostate Cancer
Journal: bioRxiv
doi: 10.1101/2021.06.04.446195
Figure Legend Snippet: There is elevated P2X4 receptor protein expression in (A) cancer compared to benign tissues (n = 456; p
Techniques Used: Expressing
Figure Legend Snippet: P2X4 purinergic receptor protein is expressed (A) modestly in the epithelium of prostate glands and more intensely on immune cells of organ donor prostates (C) modestly in benign glands and more intensely in prostatic intraepithelial neoplasia (PIN) and cancer in prostatectomy samples and (D) on immune cells. (B) H E staining confirmed tissue types. (E) Dual staining showed that tryptase positive mast cells (red) do not express P2X4 protein (brown), but some CD66 positive neutrophils (brown) and most CD68 positive macrophages (brown) express P2X4 receptors (red). Images are at 40x magnification unless otherwise stated.
Techniques Used: Staining
Figure Legend Snippet: (A, B) P2X4 mRNA expression was detected in cancer and immune cells and in various metastatic sites. (C) P2X4 mRNA expression (brown) detected on CD68 + (red) macrophages in metastatic tissues. (D) There is a positive correlation between CD68 and P2X4 mRNA expression in metastatic tissues.
Techniques Used: Expressing
Figure Legend Snippet: (A) P2X4 antagonist 5-BDBD resulted in a dose-dependent decrease in PCa cell viability. (B) P2X4 down in LNCaP cells resulted attenuated cell growth over time. (C) Treatment with 5-BDBD resulted in a dose-dependent induction of cleaved caspase-3 in PCa cells.
Techniques Used:
Figure Legend Snippet: (A) Nucleotide treatment and (B) P2X4 knock down in DU145 cells. (C) CTP treatment increased cell migration and invasion while 5-BDBD and P2X4 knock down decreased cell migration and invasion in transwell assays. (P = parental, KD = knockdown)
Techniques Used: Migration
Figure Legend Snippet: (A) Robust P2X4 mRNA expression is detected in mouse prostate tumors. (B) CRISPR knockdown of P2X4 receptor expression in Myc-CaP cells (n=10) resulted in attenuated allograft growth in FVB/NJ mice compared to control Myc-CaP cells (n=10). (C) Human c-Myc RISH and P2X4 and AR IHC analysis confirmed P2X4 reduced expression in Myc-CaP allograft tissue from mice. (D) P2X4 positive regions lacking AR and human c-Myc stain positive for CD11b and CD3 in Myc-CaP allograft tissue from mice.
Techniques Used: Expressing, CRISPR, Mouse Assay, Immunohistochemistry, Staining
13) Product Images from "Astragalin Alleviates Neuropathic Pain by Suppressing P2X4-Mediated Signaling in the Dorsal Root Ganglia of Rats"
Article Title: Astragalin Alleviates Neuropathic Pain by Suppressing P2X4-Mediated Signaling in the Dorsal Root Ganglia of Rats
Journal: Frontiers in Neuroscience
doi: 10.3389/fnins.2020.570831
Figure Legend Snippet: AST decreases P2X4 level in the dorsal root ganglia (DRG) of CCI rats. (A,B) Relative expression of P2X4 mRNA (A) and protein (B) was increased in CCI rats compared to the Ctrl group, and decreased in AST-treated CCI rats compared to CCI rats. (C) P2X4 protein expression in CCI rats was decreased by P2X4-selective antagonist 5-BDBD compared with that in the untreated CCI rats. Data are presented as mean ± SEM of three independent experiments for each bar (Each group sample was from eight rats.). ** p
Techniques Used: AST Assay, Expressing
Figure Legend Snippet: Effect of AST on ATP-activated currents in HEK293 cells expressing P2X4. (A) Representative traces showing that AST (50 μM) inhibited the ATP (100 μM)-induced current in HEK293 cells transfected with P2X4 plasmid. Control cells not transfected with P2X4 do not respond to ATP at the concentrations. (B) Histogram of current density in the presence of indicated drugs. AST (50 μM) and the P2X4-selective antagonist 5-BDBD (10 μM) inhibited the ATP-activated current in HEK293 cells. DMSO has no effect on ATP-activated current. (C) Concentration–response curve for AST obtained with 100 μM ATP. The IC 50 value 33.73 ± 2.25 μM was derived from the equation of the sigmoidal function giving the best fit to the data. Each data point represents mean ± SEM of six cells. *** p
Techniques Used: AST Assay, Expressing, Transfection, Plasmid Preparation, Concentration Assay, Derivative Assay
Figure Legend Snippet: Molecular docking of AST with hP2X4 protein. (A) Forward view. (B) Top view. (C) Chemical structure of AST. (D) Docking bag. The green and blue rod structures are the A and B chains of the hP2X4 protein, respectively; the yellow dotted line is the hydrogen bond connecting the residue on the chain and AST. The binding energy of AST with P2X4 was –7.3 kcal/mol.
Techniques Used: AST Assay, Binding Assay
Figure Legend Snippet: AST inhibits satellite glial cell (SGC) activation in DRG of CCI rats. (A) Colocalization of P2X4 and GFAP in rat DRG was detected by double immunofluorescence labeling. Green and red signals are GFAP and P2X4 labeled with fluorescein isothiocyanate and tetramethylrhodamine, respectively. Arrows indicate cells positive for both P2X4 and GFAP. Scale bar, 20 μm. (B) P2X4 and GFAP colocalization was more extensive in CCI rats than in Ctrl rats and was lower in CCI rats treated with AST than in untreated CCI rats. (C) Western blot analysis of GFAP protein level. GFAP expression was increased in CCI rats compared to Ctrl rats, an effect that was abrogated by AST treatment. Data are presented as mean ± SEM. For fluorescence analysis, eight photos from four rats were taken from each group to get the mean number. For western blotting analysis, each group samples were from eight rats, and three independent repeated experiments were conducted. *** p
Techniques Used: AST Assay, Activation Assay, Immunofluorescence, Labeling, Western Blot, Expressing, Fluorescence
Figure Legend Snippet: 5-BDBD inhibits extracellular signal-regulated kinase (ERK) signaling in DRG of CCI rats. (A) Western blot analysis of pERK, ERK, and β-actin levels. (B) Total ERK level did not differ significantly between groups. (C) pERK1/2 level was increased in CCI rats compared to Ctrl rats and decreased in CCI rats by treatment with the P2X4 antagonist 5-BDBD. Data are presented as mean ± SEM of three independent experiments for each bar (Each group sample was from eight rats.) * p
Techniques Used: Western Blot
14) Product Images from "Astragalin Alleviates Neuropathic Pain by Suppressing P2X4-Mediated Signaling in the Dorsal Root Ganglia of Rats"
Article Title: Astragalin Alleviates Neuropathic Pain by Suppressing P2X4-Mediated Signaling in the Dorsal Root Ganglia of Rats
Journal: Frontiers in Neuroscience
doi: 10.3389/fnins.2020.570831
Figure Legend Snippet: AST decreases P2X4 level in the dorsal root ganglia (DRG) of CCI rats. (A,B) Relative expression of P2X4 mRNA (A) and protein (B) was increased in CCI rats compared to the Ctrl group, and decreased in AST-treated CCI rats compared to CCI rats. (C) P2X4 protein expression in CCI rats was decreased by P2X4-selective antagonist 5-BDBD compared with that in the untreated CCI rats. Data are presented as mean ± SEM of three independent experiments for each bar (Each group sample was from eight rats.). ** p
Techniques Used: AST Assay, Expressing
Figure Legend Snippet: Effect of AST on ATP-activated currents in HEK293 cells expressing P2X4. (A) Representative traces showing that AST (50 μM) inhibited the ATP (100 μM)-induced current in HEK293 cells transfected with P2X4 plasmid. Control cells not transfected with P2X4 do not respond to ATP at the concentrations. (B) Histogram of current density in the presence of indicated drugs. AST (50 μM) and the P2X4-selective antagonist 5-BDBD (10 μM) inhibited the ATP-activated current in HEK293 cells. DMSO has no effect on ATP-activated current. (C) Concentration–response curve for AST obtained with 100 μM ATP. The IC 50 value 33.73 ± 2.25 μM was derived from the equation of the sigmoidal function giving the best fit to the data. Each data point represents mean ± SEM of six cells. *** p
Techniques Used: AST Assay, Expressing, Transfection, Plasmid Preparation, Concentration Assay, Derivative Assay
Figure Legend Snippet: Molecular docking of AST with hP2X4 protein. (A) Forward view. (B) Top view. (C) Chemical structure of AST. (D) Docking bag. The green and blue rod structures are the A and B chains of the hP2X4 protein, respectively; the yellow dotted line is the hydrogen bond connecting the residue on the chain and AST. The binding energy of AST with P2X4 was –7.3 kcal/mol.
Techniques Used: AST Assay, Binding Assay
Figure Legend Snippet: AST inhibits satellite glial cell (SGC) activation in DRG of CCI rats. (A) Colocalization of P2X4 and GFAP in rat DRG was detected by double immunofluorescence labeling. Green and red signals are GFAP and P2X4 labeled with fluorescein isothiocyanate and tetramethylrhodamine, respectively. Arrows indicate cells positive for both P2X4 and GFAP. Scale bar, 20 μm. (B) P2X4 and GFAP colocalization was more extensive in CCI rats than in Ctrl rats and was lower in CCI rats treated with AST than in untreated CCI rats. (C) Western blot analysis of GFAP protein level. GFAP expression was increased in CCI rats compared to Ctrl rats, an effect that was abrogated by AST treatment. Data are presented as mean ± SEM. For fluorescence analysis, eight photos from four rats were taken from each group to get the mean number. For western blotting analysis, each group samples were from eight rats, and three independent repeated experiments were conducted. *** p
Techniques Used: AST Assay, Activation Assay, Immunofluorescence, Labeling, Western Blot, Expressing, Fluorescence
Figure Legend Snippet: 5-BDBD inhibits extracellular signal-regulated kinase (ERK) signaling in DRG of CCI rats. (A) Western blot analysis of pERK, ERK, and β-actin levels. (B) Total ERK level did not differ significantly between groups. (C) pERK1/2 level was increased in CCI rats compared to Ctrl rats and decreased in CCI rats by treatment with the P2X4 antagonist 5-BDBD. Data are presented as mean ± SEM of three independent experiments for each bar (Each group sample was from eight rats.) * p
Techniques Used: Western Blot
15) Product Images from "Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4‐mediated calcium entry in microglia, et al. Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4‐mediated calcium entry in microglia"
Article Title: Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4‐mediated calcium entry in microglia, et al. Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4‐mediated calcium entry in microglia
Journal: Glia
doi: 10.1002/glia.23847
Figure Legend Snippet: Channel expression changes in differentially activated microglia. (a) Purinergic currents from three representative undifferentiated microglia evoked by a 3‐s pulse of either ATP or BzATP while voltage clamped at −70 mV. (b) Potentiation of ATP‐induced currents by the P2X4‐selective positive modulator ivermectin ( left ). Quantification of area under the curve (AUC) showed a 3.04 ± 0.80‐fold increase in potentiation ( n = 4) currents induced by 0.03 mM ATP by in the presence of ivermectin ( right ). Statistical significance (** p
Techniques Used: Expressing
Figure Legend Snippet: Expression changes of Kv1.3 channels and P2X4 receptors in in microglia isolated from Cx3CR1 +/EGFP transgenic mice 8 days after middle cerebral artery occlusion (MCAO) as a model of ischemic stroke. Sample immunofluorescence staining of 14‐μM thick coronal brain sections from the 6‐mm depth showing (a) increased Kv1.3 ( red ) and (b) P2X4 ( red ) immunoreactivity in ipsilateral Cx3CR1 +/EGFP ( green ) cells but not contralateral cells. Each channel was analyzed on n = 3–4 coronal sections from N = 3 male and 3 female mice. (c) P2X4 (d) Kv1.3 and (e) Kir2.1 current densities measured from CD11b + Cx3CR1 +/EGFP microglia acutely isolated from the ipsilateral hemisphere (8 days after MCAO) compared to microglia isolated from the contralateral side. Statistical significance determined by one‐way analysis of variance (ANOVA) followed by Tukey–Cramer's post hoc (alpha = 0.05). * p
Techniques Used: Expressing, Isolation, Transgenic Assay, Mouse Assay, Immunofluorescence, Staining
Figure Legend Snippet: Kv1.3 blockade depolarizes microglia and disrupts resistance to ATP‐induced membrane depolarization. (a) Kv1.3 inhibitors do not cross‐react with P2X4. Sample recording of P2X4 currents elicited by 0.1 mM ATP in a Chinese Hamster Ovary (CHO) cell at the 0, 5, and 10‐min time points displaying characteristic time‐dependent current rundown. (b) Bar graphs showing normalized current for control cells ( n = 5), PAP‐1 (1 μM) treated cells ( n = 4), and ShK‐223 (100 nM) treated cells ( n = 5). Inhibitors were added immediately after the first ATP pulse and remained in the recording chamber throughout the duration between and during subsequent ATP pulses. Error bars denote means ± SD . (c) Voltage‐clamp currents before and after inhibition of Kv1.3 with 100 nM ShK‐223 in an undifferentiated microglial cell. (g) Current‐clamp displaying ATP‐induced depolarization (AID) of resting membrane potential (RMP) before and after ShK‐223 in the same undifferentiated cell. (e) Scatterplots summarizing RMP and AMP levels before and after ShK‐223 for undifferentiated cells ( n = 14). (f) Voltage‐clamp currents before and after inhibition of Kv1.3 with 100 nM ShK‐223 in an lipopolysaccharides (LPS)‐stimulated microglial cell. (g) Current‐clamp displaying AID of RMP before and after ShK‐223 in the same LPS‐stimulated cell. (h) Scatterplots summarizing RMP and AMP levels for LPS‐treated cells ( n = 8). Statistical significance determined by paired t test. *** p
Techniques Used: Inhibition
16) Product Images from "Overexpression of Purinergic P2X4 Receptors in Hippocampus Rescues Memory Impairment in Rats with Type 2 Diabetes"
Article Title: Overexpression of Purinergic P2X4 Receptors in Hippocampus Rescues Memory Impairment in Rats with Type 2 Diabetes
Journal: Neuroscience Bulletin
doi: 10.1007/s12264-020-00478-7
Figure Legend Snippet: P2X4R expression is down-regulated in the hippocampus of T2DM rats. A Protein expression of P2X4Rs in the hippocampus of T2DM and control (CON) rats ( n = 4; *P
Techniques Used: Expressing
Figure Legend Snippet: Overexpression of P2X4Rs blocks the activation of microglia. A Representative images of AAV-P2X4R (green) and CD11b (red) staining in the hippocampus of a T2DM rat (arrows, co-localization of AAV-P2X4R and CD11b; broken lines outline the hippocampus). B P2X4R expression after microinjection of AAV-P2X4R or AAV-NC ( n = 4/group; ** *P
Techniques Used: Over Expression, Activation Assay, Staining, Expressing
Figure Legend Snippet: P2X4Rs are mainly expressed in microglia in the hippocampus. A Representative images of P2X4Rs (green) co-expressed with CD11b (red), but not with GFAP (red) or NeuN (red) (scale bar, 100 μm). B Representative images and statistics showing the percentages of P2X4R-positive cells in T2DM and control (CON) rats ( n = 4/group; *P
Techniques Used:
17) Product Images from "Minocycline Attenuates Experimental Subarachnoid Hemorrhage in Rats"
Article Title: Minocycline Attenuates Experimental Subarachnoid Hemorrhage in Rats
Journal: Open Life Sciences
doi: 10.1515/biol-2019-0067
Figure Legend Snippet: The effect of minocycline on activation of P2X4R and p38at 48 hours post SAH.a. Double fluorescence labeling of P2X4R (red) and Iba-1 (green) in the prefrontal cortex region (magnification ×400, scale bar=50 μm).b. Percentage of P2X4R-positive cells in sham, SAH+saline, SAH+low-dose minocycline (L-Min), and SAH+high-dose minocycline (H-Min) groups.c d. Protein expression of P2X4R, total p38, p-p38, and p-p38/total p38. * P
Techniques Used: Activation Assay, Fluorescence, Labeling, Expressing
18) Product Images from "Minocycline Attenuates Experimental Subarachnoid Hemorrhage in Rats"
Article Title: Minocycline Attenuates Experimental Subarachnoid Hemorrhage in Rats
Journal: Open Life Sciences
doi: 10.1515/biol-2019-0067
Figure Legend Snippet: The effect of minocycline on activation of P2X4R and p38at 48 hours post SAH.a. Double fluorescence labeling of P2X4R (red) and Iba-1 (green) in the prefrontal cortex region (magnification ×400, scale bar=50 μm).b. Percentage of P2X4R-positive cells in sham, SAH+saline, SAH+low-dose minocycline (L-Min), and SAH+high-dose minocycline (H-Min) groups.c d. Protein expression of P2X4R, total p38, p-p38, and p-p38/total p38. * P
Techniques Used: Activation Assay, Fluorescence, Labeling, Expressing
19) Product Images from "Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD"
Article Title: Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD
Journal: American Journal of Physiology - Renal Physiology
doi: 10.1152/ajprenal.00395.2019
Figure Legend Snippet: A : schema of CRISPR-induced insertion in exon 2 of the P2X receptor ( P2rx7 ) gene leading to a frameshift mutation. B : Western blot demonstrating P2X7 protein expression in total kidney samples of polycystic kidney (PCK) rat littermates with different genotypes. GAPDH expression was used as a loading control. C : P2X4 abundance in kidneys was not affected in heterozygous or knockout rats. D : body weight progression with aging ( n = 6–14). E : 24-h urinary sodium excretion measured in young and adult rats ( n = 11–21). F : glomerular filtration rate (GFR) measured in 24-wk-old Sprague-Dawley (SD), PCK. P2rx7 +/+ , and PCK. P2rx7 −/− rats ( n = 5–7). PCK. P2rx7 +/+ , PCK rats with intact P2rx7 ; PCK. P2rx7 −/− , PCK rats with P2rx7 −/− knockout; PCK. P2rx7 +/− , PCK rats heterozygous for P2rx7 −/− . Plasma FITC-inulin clearance in conscious animals was measured after a bolus intravenous administration and GFR was calculated in the 2-compartment model. * P
Techniques Used: CRISPR, Mutagenesis, Western Blot, Expressing, Knock-Out, Filtration
20) Product Images from "Generation and Characterization of Specific Monoclonal Antibodies and Nanobodies Directed Against the ATP-Gated Channel P2X4"
Article Title: Generation and Characterization of Specific Monoclonal Antibodies and Nanobodies Directed Against the ATP-Gated Channel P2X4
Journal: Frontiers in Cellular Neuroscience
doi: 10.3389/fncel.2019.00498
Figure Legend Snippet: Analysis of cloned nanobodies against mouse or human P2X4. (A) Immunofluorescence analysis of the reactivities of monovalent his6x-c-myc-tagged nanobodies with non-permeabilized CHO cells transiently co-transfected with GFP and either mouse (top panel) or human (bottom panel) P2X4 Y378F. Bound nanobodies were detected by sequential staining with a C-myc tag-specific mouse mAb followed by PE-conjugated rabbit anti-mouse IgG. Scale bar: 400 μm. (B,C) Flow cytometry analysis of the binding specificity of the different selected nanobodies. HEK cells were co-transfected with GFP and either human, mouse, or rat P2X4 Y378F. The FACS analyses in (B,C) were performed on transiently transfected HEK cells using bivalent Nb-rabbit IgG heavy chain antibodies. Bound antibodies were detected with Alexa 647-conjugated goat anti-rabbit IgG. (B) Example of results obtained with Nb 271, which binds all tested P2X4 receptors. (C) Binding specificity of the different selected nanobodies. Gray graphs correspond to control staining using only the secondary antibody. Nb isotype corresponds to a control with an unrelated isotype nanobody. The different staining protocols used to account for the stronger labeling intensity of human P2X4 transfected cells in (B,C) vs. (A) . The relative staining intensities of the nanobodies are the same in panels (A,B) , i.e., for mP2X4: 325 > 284 > 318 = 262 and for hP2X4: 262 > 318 > 284 > 325.
Techniques Used: Clone Assay, Immunofluorescence, Transfection, Staining, Flow Cytometry, Binding Assay, FACS, Labeling
Figure Legend Snippet: Biochemical properties of Nodu 246 hybridoma. (A) Recognition of denatured human and mouse P2X4 receptors by mAb Nodu 246 was assessed by Western blotting. Protein extracts from a HEK cell line stably expressing the human P2X4 and HEK cells transiently transfected with an expression construct for Flag/HA-tagged mouse P2X4 were analyzed by Western blot after electrophoresis of cell lysates on an SDS-polyacrylamide gel. Blots were incubated either with a rabbit polyclonal anti-P2X4 peptide antibody (Alomone) or with rat monoclonal antibody Nodu 246. Bound antibodies were revealed with peroxidase-conjugated anti-rabbit IgG or anti-rat IgG. The monoclonal mAb Nodu 246 evidently does not recognize the denatured forms of human and mouse P2X4 (right panel), as opposed to the rabbit polyclonal anti-peptide antibody (left panel). (B) P2X4 in mouse WT and P2X4 KO bone marrow-derived macrophage (BMDM) was specifically immunoprecipitated with Nodu 246. Immunoprecipitated proteins were separated by electrophoresis on SDS-polyacrylamide gels and immunoblotted with a polyclonal rabbit anti-P2X4 peptide antibody (Alomone). The same protein extracts were loaded on both panels. Note the higher apparent molecular weight of the Flag/HA-tagged mouse P2X4 compared to human P2X4.
Techniques Used: Western Blot, Stable Transfection, Expressing, Transfection, Construct, Electrophoresis, Incubation, Derivative Assay, Immunoprecipitation, Molecular Weight
Figure Legend Snippet: Flow cytometry analysis of mouse P2X4 expression using Nodu 246. (A) Cell surface and intracellular expression of mouse P2X4 by P2X4-transfected and non-transfected HEK cells was assessed by flow cytometry after sequential incubation of cells with rat mAb Nodu 246 followed by a secondary PE-conjugated anti-rat-IgG antibody (2nd antibody), in non-permeabilized or permeabilized cells. Similar experiments were performed with BMDM obtained from WT and P2X4 −/− mice. Cells were either non-permeabilized (B) or permeabilized (C) . Note that permeabilization greatly enhanced the detection of mouse P2X4 in BMDM, further supporting the intracellular localization of the native receptor.
Techniques Used: Flow Cytometry, Expressing, Transfection, Incubation, Mouse Assay
Figure Legend Snippet: Immunocyto- and -histochemistry with Nodu 246. (A) Co-immunostaining of WT and P2X4 −/− BMDM with Nodu 246 and the lysosomal marker CD68. Scale bar: 5 μm. (B) Co-immunostaining of WT and P2X4 −/− cultured mouse microglia using the rat-monoclonal antibody Nodu 246 and the lysosomal marker LC3b. Scale bar: 10 μm. (C) Immunostaining of dorsal root ganglion (DRG) of WT and P2X4 −/− DRG. Scale bar: 50 μm. (D) Top panel, co-immunostaining of microglial cells (green, Iba1) and P2X4 (red, Nodu 246) and amyloid plaques (blue, AmyloGlo) in the cortex of brains from APP/PS1 and APP/PS1; P2X4 −/− mice. P2X4 is localized in the intracellular compartments of activated microglia clustered around amyloid deposits. Scale bar 20 μm. Bottom panel, low magnification of the cortical region of APP/PS1 and APP/PS1; P2X4 −/− mice stained with anti-Iba1 (green) and Nodu 246 (red). Note the dim P2X4 immunostaining in cortical neurons. Scale bar 50 μm.
Techniques Used: Immunostaining, Marker, Cell Culture, Mouse Assay, Staining
Figure Legend Snippet: Flow cytometry analysis of native mouse P2X4 expressed by peritoneal mast cells and BMDM. (A) Peritoneal cells from WT and P2X4 −/− were stimulated (white) or not (gray) for 10 min with 2 mM ATP at 37°C. Cells were washed and incubated at 4°C with anti-CD11b-PerCO and anti-FcεR1-PE as well as with various antibodies directed against P2X4 and appropriate secondary antibodies (AF647 or AF488). CD107a (LAMP1-FITC) was used as a control experiment. Mast cells were identified as CD11b low, FcεR1+. mAb Nodu 246: rat monoclonal antibody; Nb 325, Nb 271: Nanobodies. (B) Similar flow cytometry experiments of WT and P2X4 −/− BMDM using Nb 271 RbhcAb. Cells were stimulated as above with 2 mM ATP (ATP, green), 3 μM ivermectin (IVM, gray), or not stimulated (nanobody, blue). Negative controls were secondary antibody only (secondary antibody, orange) and unlabeled cells (Neg, red).
Techniques Used: Flow Cytometry, Incubation
Figure Legend Snippet: Identification of P2X4-specific monoclonal antibodies. (A) Schematic diagram of the rat immunization protocol. Rats were immunized five times by trans-dermic biolistic gene delivery (six shots per immunization) with expression vectors encoding either mouse WT P2X4 (rat RG23) or mouse and human P2X4-Y378F (rat RG96). Each rat received a final injection of CHO cells expressing either mouse P2X4 or a mixture of mouse and human P2X4-Y378F. Pre-immune sera were drawn at day 0 and immune sera at day 74, prior to the final boost at day 123. Terminal bleeding was performed on day 131, followed by cell fusion and hybridoma selection. (B) Pre-immune sera, immune sera (left panels, dilution 1/1,600) of rat RG96 and hybridoma supernatants derived from RG96 (right panels) were tested for reactivity with CHO-cells transiently co-transfected with expression constructs for nuclear GFP and either human P2X4 or mouse P2X4-Y378F, as indicated. Different hybridoma show specificity toward human or mouse P2X4 receptors. Scale bar: 200 μm. (C) Specific characteristics of the different identified hybridoma.
Techniques Used: Expressing, Injection, Selection, Derivative Assay, Transfection, Construct
21) Product Images from "Generation and Characterization of Specific Monoclonal Antibodies and Nanobodies Directed Against the ATP-Gated Channel P2X4"
Article Title: Generation and Characterization of Specific Monoclonal Antibodies and Nanobodies Directed Against the ATP-Gated Channel P2X4
Journal: Frontiers in Cellular Neuroscience
doi: 10.3389/fncel.2019.00498
Figure Legend Snippet: Analysis of cloned nanobodies against mouse or human P2X4. (A) Immunofluorescence analysis of the reactivities of monovalent his6x-c-myc-tagged nanobodies with non-permeabilized CHO cells transiently co-transfected with GFP and either mouse (top panel) or human (bottom panel) P2X4 Y378F. Bound nanobodies were detected by sequential staining with a C-myc tag-specific mouse mAb followed by PE-conjugated rabbit anti-mouse IgG. Scale bar: 400 μm. (B,C) Flow cytometry analysis of the binding specificity of the different selected nanobodies. HEK cells were co-transfected with GFP and either human, mouse, or rat P2X4 Y378F. The FACS analyses in (B,C) were performed on transiently transfected HEK cells using bivalent Nb-rabbit IgG heavy chain antibodies. Bound antibodies were detected with Alexa 647-conjugated goat anti-rabbit IgG. (B) Example of results obtained with Nb 271, which binds all tested P2X4 receptors. (C) Binding specificity of the different selected nanobodies. Gray graphs correspond to control staining using only the secondary antibody. Nb isotype corresponds to a control with an unrelated isotype nanobody. The different staining protocols used to account for the stronger labeling intensity of human P2X4 transfected cells in (B,C) vs. (A) . The relative staining intensities of the nanobodies are the same in panels (A,B) , i.e., for mP2X4: 325 > 284 > 318 = 262 and for hP2X4: 262 > 318 > 284 > 325.
Techniques Used: Clone Assay, Immunofluorescence, Transfection, Staining, Flow Cytometry, Binding Assay, FACS, Labeling
Figure Legend Snippet: Biochemical properties of Nodu 246 hybridoma. (A) Recognition of denatured human and mouse P2X4 receptors by mAb Nodu 246 was assessed by Western blotting. Protein extracts from a HEK cell line stably expressing the human P2X4 and HEK cells transiently transfected with an expression construct for Flag/HA-tagged mouse P2X4 were analyzed by Western blot after electrophoresis of cell lysates on an SDS-polyacrylamide gel. Blots were incubated either with a rabbit polyclonal anti-P2X4 peptide antibody (Alomone) or with rat monoclonal antibody Nodu 246. Bound antibodies were revealed with peroxidase-conjugated anti-rabbit IgG or anti-rat IgG. The monoclonal mAb Nodu 246 evidently does not recognize the denatured forms of human and mouse P2X4 (right panel), as opposed to the rabbit polyclonal anti-peptide antibody (left panel). (B) P2X4 in mouse WT and P2X4 KO bone marrow-derived macrophage (BMDM) was specifically immunoprecipitated with Nodu 246. Immunoprecipitated proteins were separated by electrophoresis on SDS-polyacrylamide gels and immunoblotted with a polyclonal rabbit anti-P2X4 peptide antibody (Alomone). The same protein extracts were loaded on both panels. Note the higher apparent molecular weight of the Flag/HA-tagged mouse P2X4 compared to human P2X4.
Techniques Used: Western Blot, Stable Transfection, Expressing, Transfection, Construct, Electrophoresis, Incubation, Derivative Assay, Immunoprecipitation, Molecular Weight
Figure Legend Snippet: Flow cytometry analysis of mouse P2X4 expression using Nodu 246. (A) Cell surface and intracellular expression of mouse P2X4 by P2X4-transfected and non-transfected HEK cells was assessed by flow cytometry after sequential incubation of cells with rat mAb Nodu 246 followed by a secondary PE-conjugated anti-rat-IgG antibody (2nd antibody), in non-permeabilized or permeabilized cells. Similar experiments were performed with BMDM obtained from WT and P2X4 −/− mice. Cells were either non-permeabilized (B) or permeabilized (C) . Note that permeabilization greatly enhanced the detection of mouse P2X4 in BMDM, further supporting the intracellular localization of the native receptor.
Techniques Used: Flow Cytometry, Expressing, Transfection, Incubation, Mouse Assay
Figure Legend Snippet: Immunocyto- and -histochemistry with Nodu 246. (A) Co-immunostaining of WT and P2X4 −/− BMDM with Nodu 246 and the lysosomal marker CD68. Scale bar: 5 μm. (B) Co-immunostaining of WT and P2X4 −/− cultured mouse microglia using the rat-monoclonal antibody Nodu 246 and the lysosomal marker LC3b. Scale bar: 10 μm. (C) Immunostaining of dorsal root ganglion (DRG) of WT and P2X4 −/− DRG. Scale bar: 50 μm. (D) Top panel, co-immunostaining of microglial cells (green, Iba1) and P2X4 (red, Nodu 246) and amyloid plaques (blue, AmyloGlo) in the cortex of brains from APP/PS1 and APP/PS1; P2X4 −/− mice. P2X4 is localized in the intracellular compartments of activated microglia clustered around amyloid deposits. Scale bar 20 μm. Bottom panel, low magnification of the cortical region of APP/PS1 and APP/PS1; P2X4 −/− mice stained with anti-Iba1 (green) and Nodu 246 (red). Note the dim P2X4 immunostaining in cortical neurons. Scale bar 50 μm.
Techniques Used: Immunostaining, Marker, Cell Culture, Mouse Assay, Staining
Figure Legend Snippet: Flow cytometry analysis of native mouse P2X4 expressed by peritoneal mast cells and BMDM. (A) Peritoneal cells from WT and P2X4 −/− were stimulated (white) or not (gray) for 10 min with 2 mM ATP at 37°C. Cells were washed and incubated at 4°C with anti-CD11b-PerCO and anti-FcεR1-PE as well as with various antibodies directed against P2X4 and appropriate secondary antibodies (AF647 or AF488). CD107a (LAMP1-FITC) was used as a control experiment. Mast cells were identified as CD11b low, FcεR1+. mAb Nodu 246: rat monoclonal antibody; Nb 325, Nb 271: Nanobodies. (B) Similar flow cytometry experiments of WT and P2X4 −/− BMDM using Nb 271 RbhcAb. Cells were stimulated as above with 2 mM ATP (ATP, green), 3 μM ivermectin (IVM, gray), or not stimulated (nanobody, blue). Negative controls were secondary antibody only (secondary antibody, orange) and unlabeled cells (Neg, red).
Techniques Used: Flow Cytometry, Incubation
Figure Legend Snippet: Identification of P2X4-specific monoclonal antibodies. (A) Schematic diagram of the rat immunization protocol. Rats were immunized five times by trans-dermic biolistic gene delivery (six shots per immunization) with expression vectors encoding either mouse WT P2X4 (rat RG23) or mouse and human P2X4-Y378F (rat RG96). Each rat received a final injection of CHO cells expressing either mouse P2X4 or a mixture of mouse and human P2X4-Y378F. Pre-immune sera were drawn at day 0 and immune sera at day 74, prior to the final boost at day 123. Terminal bleeding was performed on day 131, followed by cell fusion and hybridoma selection. (B) Pre-immune sera, immune sera (left panels, dilution 1/1,600) of rat RG96 and hybridoma supernatants derived from RG96 (right panels) were tested for reactivity with CHO-cells transiently co-transfected with expression constructs for nuclear GFP and either human P2X4 or mouse P2X4-Y378F, as indicated. Different hybridoma show specificity toward human or mouse P2X4 receptors. Scale bar: 200 μm. (C) Specific characteristics of the different identified hybridoma.
Techniques Used: Expressing, Injection, Selection, Derivative Assay, Transfection, Construct