asic3  (Alomone Labs)


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    Structured Review

    Alomone Labs asic3
    NGF-based gene therapy alleviated mechanical hyperalgesia via downregulating <t>ASIC3.</t> a The transduction of lentivirus vector into 293T cells was successful. The left figure shows the fluorescence field. The right figure reveals the confirmation of NGF shRNA sequence through DNA sequencing. b Lentivirus vector was transduced into trigeminal neurons in trigeminal ganglia (TG) 7 days following the administration of lentivirus vector into TG. The lentivirus vector carrying NGF shRNA was successful in silencing NGF in TG ( c real-time PCR, d , e western blotting). f Lentivirus transduction alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the lenti + force group than in the vehicle + force group on days 1, 3, and 5. NGF knockdown downregulated the expression of ASIC3 in TG ( g real-time PCR; h , i western blotting). * P
    Asic3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/asic3/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    asic3 - by Bioz Stars, 2022-07
    93/100 stars

    Images

    1) Product Images from "Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3"

    Article Title: Retrograde nerve growth factor signaling modulates tooth mechanical hyperalgesia induced by orthodontic tooth movement via acid-sensing ion channel 3

    Journal: International Journal of Oral Science

    doi: 10.1038/s41368-021-00124-6

    NGF-based gene therapy alleviated mechanical hyperalgesia via downregulating ASIC3. a The transduction of lentivirus vector into 293T cells was successful. The left figure shows the fluorescence field. The right figure reveals the confirmation of NGF shRNA sequence through DNA sequencing. b Lentivirus vector was transduced into trigeminal neurons in trigeminal ganglia (TG) 7 days following the administration of lentivirus vector into TG. The lentivirus vector carrying NGF shRNA was successful in silencing NGF in TG ( c real-time PCR, d , e western blotting). f Lentivirus transduction alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the lenti + force group than in the vehicle + force group on days 1, 3, and 5. NGF knockdown downregulated the expression of ASIC3 in TG ( g real-time PCR; h , i western blotting). * P
    Figure Legend Snippet: NGF-based gene therapy alleviated mechanical hyperalgesia via downregulating ASIC3. a The transduction of lentivirus vector into 293T cells was successful. The left figure shows the fluorescence field. The right figure reveals the confirmation of NGF shRNA sequence through DNA sequencing. b Lentivirus vector was transduced into trigeminal neurons in trigeminal ganglia (TG) 7 days following the administration of lentivirus vector into TG. The lentivirus vector carrying NGF shRNA was successful in silencing NGF in TG ( c real-time PCR, d , e western blotting). f Lentivirus transduction alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the lenti + force group than in the vehicle + force group on days 1, 3, and 5. NGF knockdown downregulated the expression of ASIC3 in TG ( g real-time PCR; h , i western blotting). * P

    Techniques Used: Transduction, Plasmid Preparation, Fluorescence, shRNA, Sequencing, DNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Expressing

    A schematic illustration depicting the mechanisms of tooth-movement-induced mechanical hyperalgesia. Once applied on teeth, orthodontic force stimulates the upregulation of NGF in periodontal fibroblasts; periodontal NGF was then retrogradely transported from periodontium to trigeminal ganglia where NGF elicits mechanical hyperalgesia through ASIC3
    Figure Legend Snippet: A schematic illustration depicting the mechanisms of tooth-movement-induced mechanical hyperalgesia. Once applied on teeth, orthodontic force stimulates the upregulation of NGF in periodontal fibroblasts; periodontal NGF was then retrogradely transported from periodontium to trigeminal ganglia where NGF elicits mechanical hyperalgesia through ASIC3

    Techniques Used:

    NGF modulated tooth mechanical hyperalgesia through ASIC3. a Real-time PCR revealed that the expression level of ASIC3 mRNA was significantly higher in the NGF group than in the sham group on days 1, 3, 5, 7, and 14 and was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 7. b , c Western blotting revealed that the expression level of ASIC3 was significantly higher in the NGF on days 1, 3, 5, 7, and 14. ASIC3 expression level was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 5. d NGF aggravated while NGF neutralizing antibody alleviated tooth mechanical hyperalgesia. The threshold of biting withdrawal was significantly lower in the NGF group than in the sham group on days 1, 3, and 5 and significantly higher in the anti-NGF group than in the sham group on day 3. e APETx2 (ASIC3 antagonist) alleviated NGF-induced mechanical hyperalgesia and GMQ (ASIC3 agonist) exacerbated anti-NGF-alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the NGF + APETx2 group than in the NGF + Saline group on days 1, 3, and 5. The threshold of biting withdrawal was and significantly lower in the anti-NGF + GMQ group than in the anti-NGF + saline group on days 1 and 3. *, # P
    Figure Legend Snippet: NGF modulated tooth mechanical hyperalgesia through ASIC3. a Real-time PCR revealed that the expression level of ASIC3 mRNA was significantly higher in the NGF group than in the sham group on days 1, 3, 5, 7, and 14 and was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 7. b , c Western blotting revealed that the expression level of ASIC3 was significantly higher in the NGF on days 1, 3, 5, 7, and 14. ASIC3 expression level was significantly lower in the anti-NGF group than in the sham group on days 1, 3, and 5. d NGF aggravated while NGF neutralizing antibody alleviated tooth mechanical hyperalgesia. The threshold of biting withdrawal was significantly lower in the NGF group than in the sham group on days 1, 3, and 5 and significantly higher in the anti-NGF group than in the sham group on day 3. e APETx2 (ASIC3 antagonist) alleviated NGF-induced mechanical hyperalgesia and GMQ (ASIC3 agonist) exacerbated anti-NGF-alleviated mechanical hyperalgesia. The threshold of biting withdrawal was significantly higher in the NGF + APETx2 group than in the NGF + Saline group on days 1, 3, and 5. The threshold of biting withdrawal was and significantly lower in the anti-NGF + GMQ group than in the anti-NGF + saline group on days 1 and 3. *, # P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot

    Orthodontic tooth movement promoted co-expression of NGF and ASIC3. a Co-expression of NGF and ASIC3 in trigeminal neurons on day 3 following tooth movement. More neurons were co-expressed with NGF and ASIC3 in the force group as compared to the sham group. b The percentage of co-positive neurons among NGF-positive neurons was significantly higher in the force group than in the sham group on days 1 and 3 ( P
    Figure Legend Snippet: Orthodontic tooth movement promoted co-expression of NGF and ASIC3. a Co-expression of NGF and ASIC3 in trigeminal neurons on day 3 following tooth movement. More neurons were co-expressed with NGF and ASIC3 in the force group as compared to the sham group. b The percentage of co-positive neurons among NGF-positive neurons was significantly higher in the force group than in the sham group on days 1 and 3 ( P

    Techniques Used: Expressing

    2) Product Images from "Acid-sensing ion channel 1a contributes to the effect of extracellular acidosis on NLRP1 inflammasome activation in cortical neurons"

    Article Title: Acid-sensing ion channel 1a contributes to the effect of extracellular acidosis on NLRP1 inflammasome activation in cortical neurons

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-015-0465-7

    ASICs and BK channels are co-expressed in cortical neurons. Detection of the expression of ASICs and BK channels in cortical neurons by double-staining immunofluorescence (original magnification ×400). Nuclei were counterstained with Hoechst33258 ( blue ). ASICs and BK channels were labeled with FITC ( green ) and TRITC ( red ). There is a co-expression between ASIC1, ASIC2, ASIC3, and BK channels
    Figure Legend Snippet: ASICs and BK channels are co-expressed in cortical neurons. Detection of the expression of ASICs and BK channels in cortical neurons by double-staining immunofluorescence (original magnification ×400). Nuclei were counterstained with Hoechst33258 ( blue ). ASICs and BK channels were labeled with FITC ( green ) and TRITC ( red ). There is a co-expression between ASIC1, ASIC2, ASIC3, and BK channels

    Techniques Used: Expressing, Double Immunofluorescence Staining, Labeling

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    Alomone Labs rabbit anti asic3 serum
    Soma size distribution of knee joint afferents (A) was unimodal with a broad range of size and similar to that of <t>ASIC3-IR</t> knee joint afferents (C). CGRP-IR knee joint afferents consisted of small to medium cells with most cells smaller than 500μm 2 (B). ASIC3 and CGRP double labeled knee joint afferents were small to medium as well (D) whereas ASIC3 without CGRP-IR knee joint afferents were larger (E). There were no significant differences in soma size distribution among each group in knee joint afferents, CGRP, ASIC3 and ASIC3+CGRP. Only significant difference in soma size distribution was observed in ASIC3 without CGRP (E). Carrageenan-induced arthritis resulted in an overall shift in soma size distribution of ASIC3 without CGRP-IR knee joint afferents to the left (E). * p
    Rabbit Anti Asic3 Serum, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Soma size distribution of knee joint afferents (A) was unimodal with a broad range of size and similar to that of ASIC3-IR knee joint afferents (C). CGRP-IR knee joint afferents consisted of small to medium cells with most cells smaller than 500μm 2 (B). ASIC3 and CGRP double labeled knee joint afferents were small to medium as well (D) whereas ASIC3 without CGRP-IR knee joint afferents were larger (E). There were no significant differences in soma size distribution among each group in knee joint afferents, CGRP, ASIC3 and ASIC3+CGRP. Only significant difference in soma size distribution was observed in ASIC3 without CGRP (E). Carrageenan-induced arthritis resulted in an overall shift in soma size distribution of ASIC3 without CGRP-IR knee joint afferents to the left (E). * p

    Journal: The journal of pain : official journal of the American Pain Society

    Article Title: Acid sensing ion channel 3 expression in mice knee joint afferents and effects of carrageenan-induced arthritis

    doi: 10.1016/j.jpain.2008.10.010

    Figure Lengend Snippet: Soma size distribution of knee joint afferents (A) was unimodal with a broad range of size and similar to that of ASIC3-IR knee joint afferents (C). CGRP-IR knee joint afferents consisted of small to medium cells with most cells smaller than 500μm 2 (B). ASIC3 and CGRP double labeled knee joint afferents were small to medium as well (D) whereas ASIC3 without CGRP-IR knee joint afferents were larger (E). There were no significant differences in soma size distribution among each group in knee joint afferents, CGRP, ASIC3 and ASIC3+CGRP. Only significant difference in soma size distribution was observed in ASIC3 without CGRP (E). Carrageenan-induced arthritis resulted in an overall shift in soma size distribution of ASIC3 without CGRP-IR knee joint afferents to the left (E). * p

    Article Snippet: The primary antisera used in this study were rabbit anti-ASIC3 serum (1:500; Alomone Labs, Jerusalem, Israel) and rabbit anti-CGRP serum (1:1000; Peninsula Laboratories, SanCalros, CA).

    Techniques: Labeling

    The number of knee joint afferents (A) and the percentage of knee joint afferents immunoreactive for CGRP (B), ASIC3 (C), ASIC3+CGRP (D) and ASIC3 without CGRP (E). Values are represented as mean±SEM. Knee joint afferents immunoreactive for CGRP, ASIC3 and ASIC3+CGRP were significantly increased 24h after carrageenan-induced arthritis. * p

    Journal: The journal of pain : official journal of the American Pain Society

    Article Title: Acid sensing ion channel 3 expression in mice knee joint afferents and effects of carrageenan-induced arthritis

    doi: 10.1016/j.jpain.2008.10.010

    Figure Lengend Snippet: The number of knee joint afferents (A) and the percentage of knee joint afferents immunoreactive for CGRP (B), ASIC3 (C), ASIC3+CGRP (D) and ASIC3 without CGRP (E). Values are represented as mean±SEM. Knee joint afferents immunoreactive for CGRP, ASIC3 and ASIC3+CGRP were significantly increased 24h after carrageenan-induced arthritis. * p

    Article Snippet: The primary antisera used in this study were rabbit anti-ASIC3 serum (1:500; Alomone Labs, Jerusalem, Israel) and rabbit anti-CGRP serum (1:1000; Peninsula Laboratories, SanCalros, CA).

    Techniques:

    Fast Blue labeling (A,B,C and D) and immnohistochemistry staining for ASIC3 (E,F,G and H) and CGRP (I,J,K and L) showing DRG from ASIC3 +/+ (first and second rows) and ASIC3 -/- (third and fourth rows) mice without joint inflammation (first and third rows) and those 24h after joint inflammation (second and fourth rows). Photos in each row are the same DRG. Arrows indicate Fast Blue labeled DRG neurons. Asterisks indicate ASIC3-IR neurons. No immunoreactivity for ASIC3 was observed in ASIC3 -/-mice(G and H), while CGRP was observed in all groups. Some DRG neurons showing both ASIC3 and CGRP immunoreactivity were observed as yellow in ASIC3+CGRP merged images (M and N).

    Journal: The journal of pain : official journal of the American Pain Society

    Article Title: Acid sensing ion channel 3 expression in mice knee joint afferents and effects of carrageenan-induced arthritis

    doi: 10.1016/j.jpain.2008.10.010

    Figure Lengend Snippet: Fast Blue labeling (A,B,C and D) and immnohistochemistry staining for ASIC3 (E,F,G and H) and CGRP (I,J,K and L) showing DRG from ASIC3 +/+ (first and second rows) and ASIC3 -/- (third and fourth rows) mice without joint inflammation (first and third rows) and those 24h after joint inflammation (second and fourth rows). Photos in each row are the same DRG. Arrows indicate Fast Blue labeled DRG neurons. Asterisks indicate ASIC3-IR neurons. No immunoreactivity for ASIC3 was observed in ASIC3 -/-mice(G and H), while CGRP was observed in all groups. Some DRG neurons showing both ASIC3 and CGRP immunoreactivity were observed as yellow in ASIC3+CGRP merged images (M and N).

    Article Snippet: The primary antisera used in this study were rabbit anti-ASIC3 serum (1:500; Alomone Labs, Jerusalem, Israel) and rabbit anti-CGRP serum (1:1000; Peninsula Laboratories, SanCalros, CA).

    Techniques: Labeling, Staining, Mouse Assay

    Plasma membrane expression and oligomeric assembly of rASIC3 and rP2X3R subunits in oocytes. For biochemical analysis, Xenopus (X.) laevis oocytes expressing the indicated proteins were surface labeled with the membrane-impermeant fluorescent Cy5 dye before protein purification. The indicated proteins were purified under non-denaturing conditions from X. laevis oocytes by Ni 2+ -NTA chromatography or Strep-Tactin chromatography, as indicated, resolved by SDS-PAGE ( a ) or BN-PAGE ( b ), and visualized by Typhoon fluorescence scanning. a Overlay of the Cy5-labeled surface form (red) of the His-rASIC3-EGFP and His-rP2X3-StrepII protomers and the GFP fluorescence (green) of the His-rASIC3-EGFP protomer. The positions of molecular mass markers (in kDa) are shown on the left. b Overlay of GFP and Cy5 fluorescence of the homomeric His-rASIC3-EGFP (lanes 1–4) and homotrimeric His-rP2X3-StrepII (lanes 15–18) and the co-expressed His-rASIC3-EGFP and His-rP2X3-StrepII receptor subunits (lanes 5–14) in their native or partial SDS-denatured forms isolated by Ni 2+ -NTA (lanes 3–6, 10, 11, 15, 16) or Strep-Tactin (lanes 7–9, 12–14, 17, 18) chromatography as indicated. Co-expression of rASIC3 and rP2X3 (lanes 5–14) originates from the co-injection of the His-rASIC3-EGFP and His-rP2X3-StrepII subunit in cRNA ratios as indicated. The number of protomers included in the respective bands are exemplarily indicated. The inset shows the indicated section of the gel with enhanced GFP fluorescence to enable the visibility of the ASIC3 trimer. The schematics and labeling on the left or right margins indicate the numbers of rASIC3 or rP2X3 protomers incorporated in the respective trimeric (bold) or dimeric or monomeric protein band of the rASIC3 or rP2X3 receptor complexes, respectively

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Plasma membrane expression and oligomeric assembly of rASIC3 and rP2X3R subunits in oocytes. For biochemical analysis, Xenopus (X.) laevis oocytes expressing the indicated proteins were surface labeled with the membrane-impermeant fluorescent Cy5 dye before protein purification. The indicated proteins were purified under non-denaturing conditions from X. laevis oocytes by Ni 2+ -NTA chromatography or Strep-Tactin chromatography, as indicated, resolved by SDS-PAGE ( a ) or BN-PAGE ( b ), and visualized by Typhoon fluorescence scanning. a Overlay of the Cy5-labeled surface form (red) of the His-rASIC3-EGFP and His-rP2X3-StrepII protomers and the GFP fluorescence (green) of the His-rASIC3-EGFP protomer. The positions of molecular mass markers (in kDa) are shown on the left. b Overlay of GFP and Cy5 fluorescence of the homomeric His-rASIC3-EGFP (lanes 1–4) and homotrimeric His-rP2X3-StrepII (lanes 15–18) and the co-expressed His-rASIC3-EGFP and His-rP2X3-StrepII receptor subunits (lanes 5–14) in their native or partial SDS-denatured forms isolated by Ni 2+ -NTA (lanes 3–6, 10, 11, 15, 16) or Strep-Tactin (lanes 7–9, 12–14, 17, 18) chromatography as indicated. Co-expression of rASIC3 and rP2X3 (lanes 5–14) originates from the co-injection of the His-rASIC3-EGFP and His-rP2X3-StrepII subunit in cRNA ratios as indicated. The number of protomers included in the respective bands are exemplarily indicated. The inset shows the indicated section of the gel with enhanced GFP fluorescence to enable the visibility of the ASIC3 trimer. The schematics and labeling on the left or right margins indicate the numbers of rASIC3 or rP2X3 protomers incorporated in the respective trimeric (bold) or dimeric or monomeric protein band of the rASIC3 or rP2X3 receptor complexes, respectively

    Article Snippet: After washing cells twice with Tris-buffered saline (TBS, 0.05 M, pH 7.4), they were treated with blocking solution (TBS containing 5% fetal calf serum and 0.3% Triton X-100) and incubated overnight at 4 °C with the guinea pig anti-P2X3 antibody (1:400, Neuromics, cat. no.: GP 10108) and the rabbit anti-ASIC3 antibody (1:400, Alomone Labs, cat. no.: ASC-018).

    Techniques: Expressing, Labeling, Protein Purification, Purification, Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Fluorescence, Isolation, Injection

    Switch of reversal potentials between co-expressed rASIC3 and rP2X3Rs in CHO cells. Whole-cell patch-clamp recordings at a holding potential of −65 mV. a Current traces induced by protons (pH 6.5) and α,β-meATP (10 µM) applied individually onto CHO-rASIC3 (left panel) and CHO-rP2X3R cells (middle panel) or successively onto CHO-rASIC3/P2X3 cells (right panel). Agonist application was at holding potentials which increased stepwise from −60 to + 90 mV in 30 mV increments. Current–voltage relationships were constructed from recordings similar to those shown in ( a ) in order to determine the reversal potentials ( E rev ) ( b ). Means ± S.E.M. of the indicated number of experiments. c Measurement of E rev of protons (left panel) and of α,β-meATP (right panel) by a similar protocol as shown in ( a ) on separate populations of CHO-rASIC3/rP2X3R cells. Under these conditions, the E rev values of these two agonists were interchanged. Current–voltage relationships constructed from recordings similar to those shown in ( c ) in order to determine the E rev ( d ). Means ± S.E.M. of the indicated number of experiments. Blockade of ASIC3 channel activity by the selective antagonist APETx2 (0.1 µM) shifted the E rev of α,β-meATP back near to its original value of around 0 mV. e In CHO-rASIC3/rP2X3R cells, ramps of 200 mV duration (−90 to +90 mV) were delivered at a holding potential of −65 mV to determine the E rev of ASIC3 after a drop of the normal pH from 7.4 to 6.5. Then, a high concentration of α,β-meATP (100 µM) was applied in order to change the distribution of extra- and intracellular ions. Eventually, the E rev was re-determined after washing out α,β-meATP. Original tracing; large capacitive artifacts at the beginning and end of the ramp-induced currents were retouched. f Current–voltage curves were constructed from the indicated number of experiments similar to that shown in ( e ). The scale labels for the vertical bars were 2 nA ( a , left panel), 1 nA ( a , middle and right panels), 1 nA ( c ) and 500 pA ( e ). The scale labels for the horizontal bars were 1 s ( a , c ) and 2 s ( e )

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Switch of reversal potentials between co-expressed rASIC3 and rP2X3Rs in CHO cells. Whole-cell patch-clamp recordings at a holding potential of −65 mV. a Current traces induced by protons (pH 6.5) and α,β-meATP (10 µM) applied individually onto CHO-rASIC3 (left panel) and CHO-rP2X3R cells (middle panel) or successively onto CHO-rASIC3/P2X3 cells (right panel). Agonist application was at holding potentials which increased stepwise from −60 to + 90 mV in 30 mV increments. Current–voltage relationships were constructed from recordings similar to those shown in ( a ) in order to determine the reversal potentials ( E rev ) ( b ). Means ± S.E.M. of the indicated number of experiments. c Measurement of E rev of protons (left panel) and of α,β-meATP (right panel) by a similar protocol as shown in ( a ) on separate populations of CHO-rASIC3/rP2X3R cells. Under these conditions, the E rev values of these two agonists were interchanged. Current–voltage relationships constructed from recordings similar to those shown in ( c ) in order to determine the E rev ( d ). Means ± S.E.M. of the indicated number of experiments. Blockade of ASIC3 channel activity by the selective antagonist APETx2 (0.1 µM) shifted the E rev of α,β-meATP back near to its original value of around 0 mV. e In CHO-rASIC3/rP2X3R cells, ramps of 200 mV duration (−90 to +90 mV) were delivered at a holding potential of −65 mV to determine the E rev of ASIC3 after a drop of the normal pH from 7.4 to 6.5. Then, a high concentration of α,β-meATP (100 µM) was applied in order to change the distribution of extra- and intracellular ions. Eventually, the E rev was re-determined after washing out α,β-meATP. Original tracing; large capacitive artifacts at the beginning and end of the ramp-induced currents were retouched. f Current–voltage curves were constructed from the indicated number of experiments similar to that shown in ( e ). The scale labels for the vertical bars were 2 nA ( a , left panel), 1 nA ( a , middle and right panels), 1 nA ( c ) and 500 pA ( e ). The scale labels for the horizontal bars were 1 s ( a , c ) and 2 s ( e )

    Article Snippet: After washing cells twice with Tris-buffered saline (TBS, 0.05 M, pH 7.4), they were treated with blocking solution (TBS containing 5% fetal calf serum and 0.3% Triton X-100) and incubated overnight at 4 °C with the guinea pig anti-P2X3 antibody (1:400, Neuromics, cat. no.: GP 10108) and the rabbit anti-ASIC3 antibody (1:400, Alomone Labs, cat. no.: ASC-018).

    Techniques: Patch Clamp, Construct, Activity Assay, Concentration Assay

    Immunoreactivity, co-immunoprecipitation and membrane expression of rASIC3 and rP2X3Rs in rat DRG neurons. a ASIC3 and P2X3R immunoreactivities in rat DRG neurons cultured in the presence (upper panel) or absence (lower panel) of nerve growth factor (NGF). Hoechst (Hoe) was used to stain the cell nuclei. b Examples of immunoprecipitation (IP) of DTSSP-treated extracts of primary sensory neurons with P2X3 antibodies revealed in western blots (WB) with anti-ASIC3 antibodies. Incubation of neuronal cultures at pH 6.8 or 7.5 has no effect. ASIC3 signal is not found after immunoprecipitation with unrelated antibodies (IgG). Input ASIC3 and P2X3 contents in total extracts are also shown. β-Actin is used as gel loading control. c Examples of immunoprecipitation of P2X3 receptors (P2X3, upper panel) with ASIC3 channels in control conditions (scramble) and after P2X3 receptor silencing (siP2X3). No P2X3/ASIC3 signal was found after siP2X3 treatment. Western blot with anti-ASIC1 or anti-ASIC2 antibodies gave no signal. Pull down with unrelated antibody (IgG) gave no signal. Quality of input lysates and equal gel loading is shown (lower panel, total lysates). d Surface expression of P2X3Rs. Example of membrane protein biotinylation experiments in CHO cells transfected with plasmids encoding for ASIC3 alone or ASIC3 plus P2X3Rs (upper panel, surface). Quality of total protein extracts and controls for equal gel loading are also shown (bottom panel, total lysates). The scale labels in the right upper and lower panels of ( a ) were 20 µm

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Immunoreactivity, co-immunoprecipitation and membrane expression of rASIC3 and rP2X3Rs in rat DRG neurons. a ASIC3 and P2X3R immunoreactivities in rat DRG neurons cultured in the presence (upper panel) or absence (lower panel) of nerve growth factor (NGF). Hoechst (Hoe) was used to stain the cell nuclei. b Examples of immunoprecipitation (IP) of DTSSP-treated extracts of primary sensory neurons with P2X3 antibodies revealed in western blots (WB) with anti-ASIC3 antibodies. Incubation of neuronal cultures at pH 6.8 or 7.5 has no effect. ASIC3 signal is not found after immunoprecipitation with unrelated antibodies (IgG). Input ASIC3 and P2X3 contents in total extracts are also shown. β-Actin is used as gel loading control. c Examples of immunoprecipitation of P2X3 receptors (P2X3, upper panel) with ASIC3 channels in control conditions (scramble) and after P2X3 receptor silencing (siP2X3). No P2X3/ASIC3 signal was found after siP2X3 treatment. Western blot with anti-ASIC1 or anti-ASIC2 antibodies gave no signal. Pull down with unrelated antibody (IgG) gave no signal. Quality of input lysates and equal gel loading is shown (lower panel, total lysates). d Surface expression of P2X3Rs. Example of membrane protein biotinylation experiments in CHO cells transfected with plasmids encoding for ASIC3 alone or ASIC3 plus P2X3Rs (upper panel, surface). Quality of total protein extracts and controls for equal gel loading are also shown (bottom panel, total lysates). The scale labels in the right upper and lower panels of ( a ) were 20 µm

    Article Snippet: After washing cells twice with Tris-buffered saline (TBS, 0.05 M, pH 7.4), they were treated with blocking solution (TBS containing 5% fetal calf serum and 0.3% Triton X-100) and incubated overnight at 4 °C with the guinea pig anti-P2X3 antibody (1:400, Neuromics, cat. no.: GP 10108) and the rabbit anti-ASIC3 antibody (1:400, Alomone Labs, cat. no.: ASC-018).

    Techniques: Immunoprecipitation, Expressing, Cell Culture, Staining, Western Blot, Incubation, Transfection

    Electrophysiological interaction of native ASIC3 and P2X3Rs in rat DRG neurons. Whole-cell patch-clamp recordings at a holding potential of −65 mV. Rat cultured DRG neurons were used in all panels. Increasing concentrations of protons (pH 7.0–5.5) ( a , upper panel) or α,β-meATP (0.3–300 µM) ( a , lower panel) were applied in the presence of 10 µM capsazepine for 2 s with 2 min or 5 min intervals, respectively. DRG neurons were treated with APETx2 (0.1 µM; b , c ) or anti-NGF (6 µg/ml for 24 h; c ) while constructing concentration–response curves for protons or α,β-meATP. Means ± S.E.M. of the indicated number of experiments. * P

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Electrophysiological interaction of native ASIC3 and P2X3Rs in rat DRG neurons. Whole-cell patch-clamp recordings at a holding potential of −65 mV. Rat cultured DRG neurons were used in all panels. Increasing concentrations of protons (pH 7.0–5.5) ( a , upper panel) or α,β-meATP (0.3–300 µM) ( a , lower panel) were applied in the presence of 10 µM capsazepine for 2 s with 2 min or 5 min intervals, respectively. DRG neurons were treated with APETx2 (0.1 µM; b , c ) or anti-NGF (6 µg/ml for 24 h; c ) while constructing concentration–response curves for protons or α,β-meATP. Means ± S.E.M. of the indicated number of experiments. * P

    Article Snippet: After washing cells twice with Tris-buffered saline (TBS, 0.05 M, pH 7.4), they were treated with blocking solution (TBS containing 5% fetal calf serum and 0.3% Triton X-100) and incubated overnight at 4 °C with the guinea pig anti-P2X3 antibody (1:400, Neuromics, cat. no.: GP 10108) and the rabbit anti-ASIC3 antibody (1:400, Alomone Labs, cat. no.: ASC-018).

    Techniques: Patch Clamp, Cell Culture, Concentration Assay

    Concentration–response curves for agonists at various ASICs and P2X3Rs expressed in CHO cells. a – c Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Both types of agonists were applied at the indicated increasing concentrations for 2 s onto CHO cells transfected with rASIC3 ( a , upper panel) or rP2X3 ( a , lower panel). The intervals between individual applications were kept at 2 min (protons) or 5 min (α,β-meATP) in order to avoid the decrease of the subsequent current amplitudes by desensitization. Drug application procedures in this and all subsequent panels of this figure were identical. CHO cells expressing rASIC3 ( b ) or rP2X3 ( c ) were treated with amiloride (AMI; 10 µM) or A-31749 (10 µM) while constructing concentration–response curves. In this and all subsequent panels of this figure, means ± S.E.M. of the indicated number of experiments are shown. Broken lines represent curves obtained in response to agonist application to CHO cells containing ASIC3 or P2X3Rs only, in the absence of any additional drug. d – f CHO cells were transfected with rASIC3 and rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with amiloride (10 µM) or A-31749 (10 µM) while constructing pH- or concentration–response curves. g – i CHO cells were transfected with hASIC3 alone or together with hP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with A-31749 (10 µM) while constructing pH- or concentration–response curves. j – l CHO cells were transfected with rASIC1a or rASIC2a alone or together with rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton-induced currents through rASIC1a ( j , upper panel, k ) or rASIC2a ( j , lower panel, l )-containing receptors. The holding potential was −65 mV in all experiments. * P

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Concentration–response curves for agonists at various ASICs and P2X3Rs expressed in CHO cells. a – c Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Both types of agonists were applied at the indicated increasing concentrations for 2 s onto CHO cells transfected with rASIC3 ( a , upper panel) or rP2X3 ( a , lower panel). The intervals between individual applications were kept at 2 min (protons) or 5 min (α,β-meATP) in order to avoid the decrease of the subsequent current amplitudes by desensitization. Drug application procedures in this and all subsequent panels of this figure were identical. CHO cells expressing rASIC3 ( b ) or rP2X3 ( c ) were treated with amiloride (AMI; 10 µM) or A-31749 (10 µM) while constructing concentration–response curves. In this and all subsequent panels of this figure, means ± S.E.M. of the indicated number of experiments are shown. Broken lines represent curves obtained in response to agonist application to CHO cells containing ASIC3 or P2X3Rs only, in the absence of any additional drug. d – f CHO cells were transfected with rASIC3 and rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with amiloride (10 µM) or A-31749 (10 µM) while constructing pH- or concentration–response curves. g – i CHO cells were transfected with hASIC3 alone or together with hP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with A-31749 (10 µM) while constructing pH- or concentration–response curves. j – l CHO cells were transfected with rASIC1a or rASIC2a alone or together with rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton-induced currents through rASIC1a ( j , upper panel, k ) or rASIC2a ( j , lower panel, l )-containing receptors. The holding potential was −65 mV in all experiments. * P

    Article Snippet: After washing cells twice with Tris-buffered saline (TBS, 0.05 M, pH 7.4), they were treated with blocking solution (TBS containing 5% fetal calf serum and 0.3% Triton X-100) and incubated overnight at 4 °C with the guinea pig anti-P2X3 antibody (1:400, Neuromics, cat. no.: GP 10108) and the rabbit anti-ASIC3 antibody (1:400, Alomone Labs, cat. no.: ASC-018).

    Techniques: Concentration Assay, Patch Clamp, Transfection, Expressing

    Plasma membrane expression and oligomeric assembly of rASIC3 and rP2X3R subunits in oocytes. For biochemical analysis, Xenopus (X.) laevis oocytes expressing the indicated proteins were surface labeled with the membrane-impermeant fluorescent Cy5 dye before protein purification. The indicated proteins were purified under non-denaturing conditions from X. laevis oocytes by Ni 2+ -NTA chromatography or Strep-Tactin chromatography, as indicated, resolved by SDS-PAGE ( a ) or BN-PAGE ( b ), and visualized by Typhoon fluorescence scanning. a Overlay of the Cy5-labeled surface form (red) of the His-rASIC3-EGFP and His-rP2X3-StrepII protomers and the GFP fluorescence (green) of the His-rASIC3-EGFP protomer. The positions of molecular mass markers (in kDa) are shown on the left. b Overlay of GFP and Cy5 fluorescence of the homomeric His-rASIC3-EGFP (lanes 1–4) and homotrimeric His-rP2X3-StrepII (lanes 15–18) and the co-expressed His-rASIC3-EGFP and His-rP2X3-StrepII receptor subunits (lanes 5–14) in their native or partial SDS-denatured forms isolated by Ni 2+ -NTA (lanes 3–6, 10, 11, 15, 16) or Strep-Tactin (lanes 7–9, 12–14, 17, 18) chromatography as indicated. Co-expression of rASIC3 and rP2X3 (lanes 5–14) originates from the co-injection of the His-rASIC3-EGFP and His-rP2X3-StrepII subunit in cRNA ratios as indicated. The number of protomers included in the respective bands are exemplarily indicated. The inset shows the indicated section of the gel with enhanced GFP fluorescence to enable the visibility of the ASIC3 trimer. The schematics and labeling on the left or right margins indicate the numbers of rASIC3 or rP2X3 protomers incorporated in the respective trimeric (bold) or dimeric or monomeric protein band of the rASIC3 or rP2X3 receptor complexes, respectively

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Plasma membrane expression and oligomeric assembly of rASIC3 and rP2X3R subunits in oocytes. For biochemical analysis, Xenopus (X.) laevis oocytes expressing the indicated proteins were surface labeled with the membrane-impermeant fluorescent Cy5 dye before protein purification. The indicated proteins were purified under non-denaturing conditions from X. laevis oocytes by Ni 2+ -NTA chromatography or Strep-Tactin chromatography, as indicated, resolved by SDS-PAGE ( a ) or BN-PAGE ( b ), and visualized by Typhoon fluorescence scanning. a Overlay of the Cy5-labeled surface form (red) of the His-rASIC3-EGFP and His-rP2X3-StrepII protomers and the GFP fluorescence (green) of the His-rASIC3-EGFP protomer. The positions of molecular mass markers (in kDa) are shown on the left. b Overlay of GFP and Cy5 fluorescence of the homomeric His-rASIC3-EGFP (lanes 1–4) and homotrimeric His-rP2X3-StrepII (lanes 15–18) and the co-expressed His-rASIC3-EGFP and His-rP2X3-StrepII receptor subunits (lanes 5–14) in their native or partial SDS-denatured forms isolated by Ni 2+ -NTA (lanes 3–6, 10, 11, 15, 16) or Strep-Tactin (lanes 7–9, 12–14, 17, 18) chromatography as indicated. Co-expression of rASIC3 and rP2X3 (lanes 5–14) originates from the co-injection of the His-rASIC3-EGFP and His-rP2X3-StrepII subunit in cRNA ratios as indicated. The number of protomers included in the respective bands are exemplarily indicated. The inset shows the indicated section of the gel with enhanced GFP fluorescence to enable the visibility of the ASIC3 trimer. The schematics and labeling on the left or right margins indicate the numbers of rASIC3 or rP2X3 protomers incorporated in the respective trimeric (bold) or dimeric or monomeric protein band of the rASIC3 or rP2X3 receptor complexes, respectively

    Article Snippet: Cell extracts were immunopurified with a rabbit anti-P2X3 antibody (Santa Cruz Biotechnology, cat. no.: sc-25694) and immunoblotted with rabbit anti-ASIC1, rabbit anti-ASIC2, rabbit anti-ASIC3, or rabbit anti-P2X3 antibodies (Alomone Labs, cat. nos.

    Techniques: Expressing, Labeling, Protein Purification, Purification, Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Fluorescence, Isolation, Injection

    Switch of reversal potentials between co-expressed rASIC3 and rP2X3Rs in CHO cells. Whole-cell patch-clamp recordings at a holding potential of −65 mV. a Current traces induced by protons (pH 6.5) and α,β-meATP (10 µM) applied individually onto CHO-rASIC3 (left panel) and CHO-rP2X3R cells (middle panel) or successively onto CHO-rASIC3/P2X3 cells (right panel). Agonist application was at holding potentials which increased stepwise from −60 to + 90 mV in 30 mV increments. Current–voltage relationships were constructed from recordings similar to those shown in ( a ) in order to determine the reversal potentials ( E rev ) ( b ). Means ± S.E.M. of the indicated number of experiments. c Measurement of E rev of protons (left panel) and of α,β-meATP (right panel) by a similar protocol as shown in ( a ) on separate populations of CHO-rASIC3/rP2X3R cells. Under these conditions, the E rev values of these two agonists were interchanged. Current–voltage relationships constructed from recordings similar to those shown in ( c ) in order to determine the E rev ( d ). Means ± S.E.M. of the indicated number of experiments. Blockade of ASIC3 channel activity by the selective antagonist APETx2 (0.1 µM) shifted the E rev of α,β-meATP back near to its original value of around 0 mV. e In CHO-rASIC3/rP2X3R cells, ramps of 200 mV duration (−90 to +90 mV) were delivered at a holding potential of −65 mV to determine the E rev of ASIC3 after a drop of the normal pH from 7.4 to 6.5. Then, a high concentration of α,β-meATP (100 µM) was applied in order to change the distribution of extra- and intracellular ions. Eventually, the E rev was re-determined after washing out α,β-meATP. Original tracing; large capacitive artifacts at the beginning and end of the ramp-induced currents were retouched. f Current–voltage curves were constructed from the indicated number of experiments similar to that shown in ( e ). The scale labels for the vertical bars were 2 nA ( a , left panel), 1 nA ( a , middle and right panels), 1 nA ( c ) and 500 pA ( e ). The scale labels for the horizontal bars were 1 s ( a , c ) and 2 s ( e )

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Switch of reversal potentials between co-expressed rASIC3 and rP2X3Rs in CHO cells. Whole-cell patch-clamp recordings at a holding potential of −65 mV. a Current traces induced by protons (pH 6.5) and α,β-meATP (10 µM) applied individually onto CHO-rASIC3 (left panel) and CHO-rP2X3R cells (middle panel) or successively onto CHO-rASIC3/P2X3 cells (right panel). Agonist application was at holding potentials which increased stepwise from −60 to + 90 mV in 30 mV increments. Current–voltage relationships were constructed from recordings similar to those shown in ( a ) in order to determine the reversal potentials ( E rev ) ( b ). Means ± S.E.M. of the indicated number of experiments. c Measurement of E rev of protons (left panel) and of α,β-meATP (right panel) by a similar protocol as shown in ( a ) on separate populations of CHO-rASIC3/rP2X3R cells. Under these conditions, the E rev values of these two agonists were interchanged. Current–voltage relationships constructed from recordings similar to those shown in ( c ) in order to determine the E rev ( d ). Means ± S.E.M. of the indicated number of experiments. Blockade of ASIC3 channel activity by the selective antagonist APETx2 (0.1 µM) shifted the E rev of α,β-meATP back near to its original value of around 0 mV. e In CHO-rASIC3/rP2X3R cells, ramps of 200 mV duration (−90 to +90 mV) were delivered at a holding potential of −65 mV to determine the E rev of ASIC3 after a drop of the normal pH from 7.4 to 6.5. Then, a high concentration of α,β-meATP (100 µM) was applied in order to change the distribution of extra- and intracellular ions. Eventually, the E rev was re-determined after washing out α,β-meATP. Original tracing; large capacitive artifacts at the beginning and end of the ramp-induced currents were retouched. f Current–voltage curves were constructed from the indicated number of experiments similar to that shown in ( e ). The scale labels for the vertical bars were 2 nA ( a , left panel), 1 nA ( a , middle and right panels), 1 nA ( c ) and 500 pA ( e ). The scale labels for the horizontal bars were 1 s ( a , c ) and 2 s ( e )

    Article Snippet: Cell extracts were immunopurified with a rabbit anti-P2X3 antibody (Santa Cruz Biotechnology, cat. no.: sc-25694) and immunoblotted with rabbit anti-ASIC1, rabbit anti-ASIC2, rabbit anti-ASIC3, or rabbit anti-P2X3 antibodies (Alomone Labs, cat. nos.

    Techniques: Patch Clamp, Construct, Activity Assay, Concentration Assay

    Immunoreactivity, co-immunoprecipitation and membrane expression of rASIC3 and rP2X3Rs in rat DRG neurons. a ASIC3 and P2X3R immunoreactivities in rat DRG neurons cultured in the presence (upper panel) or absence (lower panel) of nerve growth factor (NGF). Hoechst (Hoe) was used to stain the cell nuclei. b Examples of immunoprecipitation (IP) of DTSSP-treated extracts of primary sensory neurons with P2X3 antibodies revealed in western blots (WB) with anti-ASIC3 antibodies. Incubation of neuronal cultures at pH 6.8 or 7.5 has no effect. ASIC3 signal is not found after immunoprecipitation with unrelated antibodies (IgG). Input ASIC3 and P2X3 contents in total extracts are also shown. β-Actin is used as gel loading control. c Examples of immunoprecipitation of P2X3 receptors (P2X3, upper panel) with ASIC3 channels in control conditions (scramble) and after P2X3 receptor silencing (siP2X3). No P2X3/ASIC3 signal was found after siP2X3 treatment. Western blot with anti-ASIC1 or anti-ASIC2 antibodies gave no signal. Pull down with unrelated antibody (IgG) gave no signal. Quality of input lysates and equal gel loading is shown (lower panel, total lysates). d Surface expression of P2X3Rs. Example of membrane protein biotinylation experiments in CHO cells transfected with plasmids encoding for ASIC3 alone or ASIC3 plus P2X3Rs (upper panel, surface). Quality of total protein extracts and controls for equal gel loading are also shown (bottom panel, total lysates). The scale labels in the right upper and lower panels of ( a ) were 20 µm

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Immunoreactivity, co-immunoprecipitation and membrane expression of rASIC3 and rP2X3Rs in rat DRG neurons. a ASIC3 and P2X3R immunoreactivities in rat DRG neurons cultured in the presence (upper panel) or absence (lower panel) of nerve growth factor (NGF). Hoechst (Hoe) was used to stain the cell nuclei. b Examples of immunoprecipitation (IP) of DTSSP-treated extracts of primary sensory neurons with P2X3 antibodies revealed in western blots (WB) with anti-ASIC3 antibodies. Incubation of neuronal cultures at pH 6.8 or 7.5 has no effect. ASIC3 signal is not found after immunoprecipitation with unrelated antibodies (IgG). Input ASIC3 and P2X3 contents in total extracts are also shown. β-Actin is used as gel loading control. c Examples of immunoprecipitation of P2X3 receptors (P2X3, upper panel) with ASIC3 channels in control conditions (scramble) and after P2X3 receptor silencing (siP2X3). No P2X3/ASIC3 signal was found after siP2X3 treatment. Western blot with anti-ASIC1 or anti-ASIC2 antibodies gave no signal. Pull down with unrelated antibody (IgG) gave no signal. Quality of input lysates and equal gel loading is shown (lower panel, total lysates). d Surface expression of P2X3Rs. Example of membrane protein biotinylation experiments in CHO cells transfected with plasmids encoding for ASIC3 alone or ASIC3 plus P2X3Rs (upper panel, surface). Quality of total protein extracts and controls for equal gel loading are also shown (bottom panel, total lysates). The scale labels in the right upper and lower panels of ( a ) were 20 µm

    Article Snippet: Cell extracts were immunopurified with a rabbit anti-P2X3 antibody (Santa Cruz Biotechnology, cat. no.: sc-25694) and immunoblotted with rabbit anti-ASIC1, rabbit anti-ASIC2, rabbit anti-ASIC3, or rabbit anti-P2X3 antibodies (Alomone Labs, cat. nos.

    Techniques: Immunoprecipitation, Expressing, Cell Culture, Staining, Western Blot, Incubation, Transfection

    Electrophysiological interaction of native ASIC3 and P2X3Rs in rat DRG neurons. Whole-cell patch-clamp recordings at a holding potential of −65 mV. Rat cultured DRG neurons were used in all panels. Increasing concentrations of protons (pH 7.0–5.5) ( a , upper panel) or α,β-meATP (0.3–300 µM) ( a , lower panel) were applied in the presence of 10 µM capsazepine for 2 s with 2 min or 5 min intervals, respectively. DRG neurons were treated with APETx2 (0.1 µM; b , c ) or anti-NGF (6 µg/ml for 24 h; c ) while constructing concentration–response curves for protons or α,β-meATP. Means ± S.E.M. of the indicated number of experiments. * P

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Electrophysiological interaction of native ASIC3 and P2X3Rs in rat DRG neurons. Whole-cell patch-clamp recordings at a holding potential of −65 mV. Rat cultured DRG neurons were used in all panels. Increasing concentrations of protons (pH 7.0–5.5) ( a , upper panel) or α,β-meATP (0.3–300 µM) ( a , lower panel) were applied in the presence of 10 µM capsazepine for 2 s with 2 min or 5 min intervals, respectively. DRG neurons were treated with APETx2 (0.1 µM; b , c ) or anti-NGF (6 µg/ml for 24 h; c ) while constructing concentration–response curves for protons or α,β-meATP. Means ± S.E.M. of the indicated number of experiments. * P

    Article Snippet: Cell extracts were immunopurified with a rabbit anti-P2X3 antibody (Santa Cruz Biotechnology, cat. no.: sc-25694) and immunoblotted with rabbit anti-ASIC1, rabbit anti-ASIC2, rabbit anti-ASIC3, or rabbit anti-P2X3 antibodies (Alomone Labs, cat. nos.

    Techniques: Patch Clamp, Cell Culture, Concentration Assay

    Concentration–response curves for agonists at various ASICs and P2X3Rs expressed in CHO cells. a – c Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Both types of agonists were applied at the indicated increasing concentrations for 2 s onto CHO cells transfected with rASIC3 ( a , upper panel) or rP2X3 ( a , lower panel). The intervals between individual applications were kept at 2 min (protons) or 5 min (α,β-meATP) in order to avoid the decrease of the subsequent current amplitudes by desensitization. Drug application procedures in this and all subsequent panels of this figure were identical. CHO cells expressing rASIC3 ( b ) or rP2X3 ( c ) were treated with amiloride (AMI; 10 µM) or A-31749 (10 µM) while constructing concentration–response curves. In this and all subsequent panels of this figure, means ± S.E.M. of the indicated number of experiments are shown. Broken lines represent curves obtained in response to agonist application to CHO cells containing ASIC3 or P2X3Rs only, in the absence of any additional drug. d – f CHO cells were transfected with rASIC3 and rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with amiloride (10 µM) or A-31749 (10 µM) while constructing pH- or concentration–response curves. g – i CHO cells were transfected with hASIC3 alone or together with hP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with A-31749 (10 µM) while constructing pH- or concentration–response curves. j – l CHO cells were transfected with rASIC1a or rASIC2a alone or together with rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton-induced currents through rASIC1a ( j , upper panel, k ) or rASIC2a ( j , lower panel, l )-containing receptors. The holding potential was −65 mV in all experiments. * P

    Journal: Nature Communications

    Article Title: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel

    doi: 10.1038/s41467-018-03728-5

    Figure Lengend Snippet: Concentration–response curves for agonists at various ASICs and P2X3Rs expressed in CHO cells. a – c Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Both types of agonists were applied at the indicated increasing concentrations for 2 s onto CHO cells transfected with rASIC3 ( a , upper panel) or rP2X3 ( a , lower panel). The intervals between individual applications were kept at 2 min (protons) or 5 min (α,β-meATP) in order to avoid the decrease of the subsequent current amplitudes by desensitization. Drug application procedures in this and all subsequent panels of this figure were identical. CHO cells expressing rASIC3 ( b ) or rP2X3 ( c ) were treated with amiloride (AMI; 10 µM) or A-31749 (10 µM) while constructing concentration–response curves. In this and all subsequent panels of this figure, means ± S.E.M. of the indicated number of experiments are shown. Broken lines represent curves obtained in response to agonist application to CHO cells containing ASIC3 or P2X3Rs only, in the absence of any additional drug. d – f CHO cells were transfected with rASIC3 and rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with amiloride (10 µM) or A-31749 (10 µM) while constructing pH- or concentration–response curves. g – i CHO cells were transfected with hASIC3 alone or together with hP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton and α,β-meATP-induced currents. Cells were treated with A-31749 (10 µM) while constructing pH- or concentration–response curves. j – l CHO cells were transfected with rASIC1a or rASIC2a alone or together with rP2X3 cDNA in a 1:1 ratio. Whole-cell patch-clamp recordings of proton-induced currents through rASIC1a ( j , upper panel, k ) or rASIC2a ( j , lower panel, l )-containing receptors. The holding potential was −65 mV in all experiments. * P

    Article Snippet: Cell extracts were immunopurified with a rabbit anti-P2X3 antibody (Santa Cruz Biotechnology, cat. no.: sc-25694) and immunoblotted with rabbit anti-ASIC1, rabbit anti-ASIC2, rabbit anti-ASIC3, or rabbit anti-P2X3 antibodies (Alomone Labs, cat. nos.

    Techniques: Concentration Assay, Patch Clamp, Transfection, Expressing