asic3  (Alomone Labs)

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

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:

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

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

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

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

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

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

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: Schematic diagram of the ASIC3-M-CSF-M2 macrophage-positive feedback loop in skin fibrosis pathogenesis. Local injury leads to the formation of an acidic microenvironment, which activates fibroblast ASIC3 receptors and promotes the increase of M-CSF secretion through the PI3K/AKT signaling pathway, leading to the polarization of M2 macrophages and increasing the secretion of TGF-β1, which mediates fibroblasts to myofibroblast differentiation.

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques:

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: Activation of ASIC3 induces polarization of macrophages to M2 phenotype in vivo. A Schematic diagram of establishing rabbit ear hypertrophic scar model and experimental plan. B Immunohistochemistry analysis of ASIC3 expression in rabbit ear tissue 21 days after wounding. Scale bars, 100 μm. C Immunofluorescence staining of rabbit ear tissue 21 days after wounding showing the total (CD68+, green) and M2 subtype (CD206+, red) macrophages after treatment by PBS, DMSO, or GMQ. Scale bars, 100 μm. D , E MFI quantification of CD68 and CD206 in (C) ( n = 3). F , G Western blot and RT-qPCR analysis of rabbit ear tissue 21 days after wounding. Total CD206 protein levels and relative mRNA levels expression in control, vehicle, and GMQ groups ( n = 3). H , I Relative mRNA expressions of M1 macrophage markers (iNOS, TNF-α) as evaluated by RT-qPCR ( n = 3). Data are expressed as the means ± SD. * P

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: Activation Assay, In Vivo, Immunohistochemistry, Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: M2 macrophage-derived TGF-β1 induces fibroblast-to-myofibroblast differentiation. A Schematic diagram of the indirect co-culture cell model and experimental plan. B ELISA results of cytokines secreted by macrophages after ASIC3 activation by the GMQ ASIC3 activator ( n = 3). C , D Fibroblasts were treated with neutralizing antibody and evaluated for α-SMA and collagen I expression ( n = 3). E , F The contractile activities of fibroblasts treated as indicated were analyzed using fibroblast-populated collagen lattice contraction after 48 h of co-culture ( n = 3). Data are expressed as the means ± SD. * P

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: Derivative Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Expressing

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: Activation of ASIC3 induces release of M-CSF from fibroblasts. A Experimental scheme of the cytokine antibody array. B The 80 cytokine antibody array was used to examine the expression of cytokines in cell supernatant. C Array results were analyzed by KEGG pathway enrichment analysis. D Semi-quantitative analysis of the expressions of MCP-1, M-CSF, MIF, OPN, OPG, and TIMP-2 presented by heat map. E MCP-1, M-CSF, MIF, OPN, OPG, and TIMP-2 expressions were determined by ELISA in cells treated as indicated for 48 h. Data are expressed as the means ± SD. * P

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: Activation Assay, Ab Array, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: ASIC3 mediates induces the release of M-CSF from fibroblasts via the PI3K/AKT/M-CSF pathway. A KEGG pathway enrichment analysis. The top 20 KEGG pathways are shown. The size of the dot reflects the gene number; the dot color indicates the q -value . B – D Western blotting and quantitative analyses of the phosphorylation of PI3K and AKT in cells treated as indicated ( n = 3). E RT-qPCR analysis of M-CSF mRNA in fibroblasts treated for 48 h with co-culture ( n = 3). F ELISA of M-CSF expression in fibroblasts treated for 48 h with co-culture ( n = 3). Data are expressed as the means ± SD. * P

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: Western Blot, Quantitative RT-PCR, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: ASIC3 is highly expressed in human hypertrophic scar/keloid tissues and human primary cells. A Schematic diagram of sample source and experimental analysis. B Representative H E staining images of human normal skin, hypertrophic scar (HS), and keloid. Scale bars, 100 μm. C Immunohistochemistry of ASIC3 expression in normal skin, hypertrophic scar and keloid. Scale bars, 100 μm and 50 μm. D Immunofluorescence analysis of ASIC3 (red) and vimentin (green) expression in human normal skin, hypertrophic scar, and keloid. Scale bars, 100 μm and 50 μm. E Western blot analysis of ASIC3 expression in normal skin, hypertrophic scar, and keloid. GAPDH served as loading control ( n = 3). F Immunofluorescence analysis of ASIC3 (red) and vimentin (green) expressions in fibroblasts derived from normal skin, hypertrophic scar, and keloid. Scale bars, 50 μm. G MFI quantification of ASIC3 in ( F ) ( n = 3). Data are expressed as the means ± SD. ** P

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: Staining, Immunohistochemistry, Expressing, Immunofluorescence, Western Blot, Derivative Assay

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: ASIC3 promotes scar formation in vivo. A Schematic diagram of the rabbit ear hypertrophic scar model. The model was subcutaneously administered either GMQ or DMSO. B Representative H E staining images of rabbit ear tissue at day 21 after wounding. Scale bars, 100 μm. The black boxes in the upper panel are enlarged in the lower panel. Scale bars, 50 μm. C The scar elevation index (SEI) was significantly higher in the GMQ group than in the vehicle and control groups ( n = 3). D Masson staining of rabbit ear tissue at 21 days after wounding. Scale bars, 100 μm. The black boxes in the upper panel were enlarged in the lower panel. Scale bars, 50 μm. E Immunofluorescence analysis of α-SMA (red) and collagen I (green) expressions in rabbit ear tissue 21 days after wounding ( n = 3). The dotted line indicates the epidermis. Scale bars, 100 μm. F – G Quantification of the α-SMA- and collagen I–positive areas by immunofluorescence. H – J Western blot analysis of α-SMA and collagen I expression at 7 days, 14 days, and 21 days after wounding. GAPDH served as a loading control. Data are expressed as the means ± SD ( n = 3). * P

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: In Vivo, Staining, Immunofluorescence, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: Activation of ASIC3 induces polarization of M0 macrophages to M2 macrophages in vitro. A Experimental design of fibroblast and macrophage indirect co-culture assay. B Representative images of THP-1 cells and 100 ng/ml PMA-derived M0 macrophages. C Flow cytometric analysis of CD68 in THP-1 cells and 100 ng/ml PMA-derived M0 macrophages. D Immunofluorescence staining of CD206 (green) in experimental and control macrophages after 48 h of co-cultivation. Scale bars, 100 μm. E , F Western blot and RT-qPCR analysis of CD206 protein levels and relative mRNA levels expression in the indicated groups ( n = 3). GAPDH served as a loading control. G Expressions of CD68 and CD206 were determined by flow cytometry. H Quantification of the flow cytometry assay results (n = 3). Data are expressed as the means ± SD. * P

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: Activation Assay, In Vitro, Co-culture Assay, Derivative Assay, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Expressing, Flow Cytometry

Journal: Cell Death & Disease

Article Title: The ASIC3-M-CSF-M2 macrophage-positive feedback loop modulates fibroblast-to-myofibroblast differentiation in skin fibrosis pathogenesis

doi: 10.1038/s41419-022-04981-9

Figure Lengend Snippet: Activation of ASIC3 promotes fibroblast-to-myofibroblast differentiation through a process requiring macrophages. A Indirect co-culture cell model and experimental plan. B Immunofluorescence staining of α-SMA (red) and collagen I (green) in fibroblasts and control after 48 h of co-cultivation. Scale bars, 100 μm. C Western blot analysis of α-SMA in the indicated groups ( n = 3). GAPDH served as a loading control. D , E α-SMA and collagen I mRNA levels after 48 h of co-cultivation ( n = 3). F – G Wound-healing assays of fibroblast migration at 12 or 24 h after wounding ( n = 3). Scale bars are 100 μm. H , I The contractile activities of fibroblasts were analyzed using fibroblast-populated collagen lattice (FPCL); images were obtained at 0, 24, and 48 h ( n = 3). Control is untreated cells. Data are expressed as the means ± SD. * P

Article Snippet: PVDF membranes were blocked and then incubated with polyclonal antibodies against ASIC3 (Alomone Labs, Israel, ASC-018), α-SMA (CST, USA, D4K9N), Collagen I (Abcam, USA, ab138492), CD206 (Novus Biologicals, USA, MAB2534), PI3K (Abcam, USA, ab191606), p-PI3K (Abcam, USA, ab182651), AKT (Abcam, USA, ab179463), p-AKT (Abcam, USA, ab38449), or GAPDH (Abcam, USA, ab8245) overnight at 4 °C and then incubated with horseradish peroxidase-conjugated secondary antibody (Abcam, USA, 7074) at room temperature for 1 h. GAPDH served as an internal control for protein normalization.

Techniques: Activation Assay, Co-Culture Assay, Immunofluorescence, Staining, Western Blot, Migration

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

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

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

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

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