asic1a polyclonal antibody  (Proteintech)

Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A Representative immunofluorescence image for ASIC1a (red) in the PVN from a naïve mouse. Scale bar, 100 μm. B Schematic of bilateral cannula injection sites into the paraventricular nucleus (PVN) (coordinates: anterior-posterior = –0.79, medial-lateral = ±0.26, dorsal-ventral = –4.35). C Schematic of pharmacological experimental procedure. D The bar graph illustrates the behavioral analysis, showing that the PcTx1 group exhibited a significant increase in the time spent in the center of the open-field test (OFT) compared to the ACSF group (n = 7 per group). * p < 0.05 (unpaired t -test). E The bar graph illustrates the behavioral analysis, showing that the PcTx1 group exhibited a significant increase in the time spent in the open arms of the elevated plus maze (EPM) compared to the ACSF group (n = 7 per group). * p < 0.05 (unpaired t -test). F The bar graph illustrates the results of the tail suspension test (TST), showing that the PcTx1 group exhibited a significant reduction in immobility time compared to the ACSF group (n = 7 per group). * p < 0.05 (unpaired t-test). G The bar graph illustrates the results of the forced swimming test (FST), showing that the PcTx1 group exhibited a significant reduction in immobility time compared to the ACSF group (n = 7 per group). * p < 0.05 (unpaired t -test). H Experimental paradigm for blood collection after forced swimming. I The bar graph illustrates plasma ACTH levels in the PcTx1 group and ACSF group following stress stimulation (n = 6 per group). * p < 0.05 (unpaired t-test). J The bar graph illustrates plasma corticosterone levels in the PcTx1 group and ACSF group following stress stimulation (n = 6 per group). * p < 0.05 (unpaired t-test). See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Immunofluorescence, Injection, Suspension, Clinical Proteomics

Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A Experimental paradigm for viral injection of AAV-DIO-mCherry in Crh-Cre mice. B, C Representative images ( B ) and statistical data ( C ) showing ASIC1a (green) neurons in the PVN co-localized with CRH (red) neurons. Scale bars, 50 μm. D Experimental paradigm for viral injection of AAV-DIO-shRNA in Crh-Cre mice. E Schematic illustration of the construction strategy for ASIC1a knockdown. F, G Representative Western blot images ( F ) and quantification ( G ) demonstrating that AAV-DIO-shASIC1a injection reduced ASIC1a expression in the PVN (n = 6 per group). * p < 0.05 (unpaired t-test). H Immunofluorescence images showing mCherry-positive neurons co-labeled with ASIC1a in the AAV-mCherry and AAV-shASIC1a groups. Scale bars, 50 μm. See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Injection, shRNA, Knockdown, Western Blot, Expressing, Immunofluorescence, Labeling

Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A-D Behavioral effects of the genetic knockdown of ASIC1a in CRH PVN neurons. Figure 3A shows the open-field test (OFT), Fig. 3B shows the elevated plus maze (EPM), Fig. 3C shows the tail suspension test (TST), and Fig. 3D shows the forced swimming test (FST), demonstrating significant behavioral differences between the AAV-mCherry group and the AAV-shASIC1a group (n = 10 per group). * p < 0.05, ** p < 0.01 (unpaired t-test). E Experimental paradigm for blood collection following forced swimming. F, G Plasma ACTH levels ( F ) and corticosterone levels ( G ) were significantly reduced in the AAV-shASIC1a group compared to the AAV-mCherry group after forced swimming (n = 6 per group). * p < 0.05, ** p < 0.01 (unpaired t-test). H Schematic of the real-time optical fiber photometry assay. I Experimental paradigm for viral injection of AAV-shRNA and AAV-DIO-GCaMP6s in Crh-Cre mice. J, K Representative images ( J ) and statistical data ( K ) showing AAV-shRNA and AAV-DIO-GCaMP6s expression in the PVN. Scale bars, 50 μm. L-O Calcium activity analysis of CRH PVN neurons in the AAV-shASIC1a group compared to the AAV-mCherry group. Figure 3L shows average calcium activity, Fig. 3M shows the area under the curve, Fig. 3N shows the maximum peak value, and Fig. 3O shows calcium signals at specific time points (n = 5 per group). * p < 0.05, ** p < 0.01 (unpaired t-test and two-way ANOVA). See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Knockdown, Suspension, Clinical Proteomics, Injection, shRNA, Expressing, Activity Assay

Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A The heatmap shows the release levels of CRH in BE (2)-C cell culture supernatants detected by ELISA. The BE (2)-C cells were treated with HBSS with a pH of 6.5 for 5 min or pretreated with 100 nM PcTx1 or 200 nM amiloride for 5 min and maintained in HBSS pH 6.5. The supernatants were collected at 1, 15, 30, 60, 90, 120, 180 min, 12 h, and 24 h for detection. The experiment was conducted with three independent biological replicates, ensuring the presence of the drug throughout both the culture and acid treatment stages (n = 3 per group). * p < 0.05, *** p < 0.001, **** p < 0.0001 (two-way ANOVA). B The bar graph shows CRH release levels in neuronal culture supernatants detected by ELISA. The experimental treatments were conducted as in Fig. 4A, and supernatants were collected at 3-h time point for detection (n = 3 per group). ** p < 0.01 (one-way ANOVA). C The bar graph shows CRH levels in the supernatant of ASIC1a knockdown neuronal culture medium detected by ELISA after HBSS pH 6.5 treatment and the supernatants were collected at 3-h time point for detection (n = 3 per group). * p < 0.05 (unpaired t- test). D Representative confocal images (scale bar = 5 μm) of CRH in BE (2)-C cells overexpressing GFP/ASIC1a-GFP for 48 h, before or 30 min after treatment with HBSS at pH 6.5. E The bar graph illustrates the relative fluorescence intensity of CRH shown in Fig. 4D (n = 3 per group). * p < 0.05, **** p < 0.0001 (one-way ANOVA). F Representative western blot assay showing the protein expression levels of CRH in primary hypothalamic neurons. Experimental treatments were conducted as in Fig. 4A, and the cells were detected at the 3-h time point. G The bar graph illustrates the grayscale scanning analysis of Fig. 4F, showing the relative expression levels of CRH (n = 3 per group). ** p < 0.01 (one-way ANOVA). H The bar graph shows the mRNA expression levels of CRH in primary hypothalamic neurons detected by qPCR. Experimental treatments were conducted as in Fig. 4F (n = 3 per group). **** p < 0.0001 (one-way ANOVA). See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Knockdown, Fluorescence, Western Blot, Expressing

Journal: Translational Psychiatry

Article Title: The acid-sensing ion channel 1a modulates anxiety- and depression-related behaviors via its influencing on the activity of corticotropin-releasing hormone-expressing neurons in the hypothalamic paraventricular nucleus in male mice

doi: 10.1038/s41398-026-03946-2

Figure Lengend Snippet: A Acid (pH 6.5)-induced changes in cytosolic Ca 2+ signal, indicated by GCaMP6 fluorescence, in cultured mouse hypothalamic neurons (n = 77 in Vector group, n = 63 in OE ASIC1a group, n = 48 in shASIC1a group, n = 73 in OE ASIC1a + PcTx1 group, n = 60 in OE ASIC1a + Amiloride group). B Representative western blot assay showing the expression of calcium signaling pathway-related proteins in primary hypothalamic neurons. Experimental treatments were conducted as in Fig. 4F. C The bar graph illustrates the grayscale scanning analysis of Fig. 5B, showing the relative expression levels of P-CaMKII (n = 3 per group). ** p < 0.01 (one-way ANOVA). D The bar graph illustrates the grayscale scanning analysis of Fig. 5B, showing the relative expression levels of c-Fos (n = 3 per group). * p < 0.05 (one-way ANOVA). E The bar graphs show the mRNA expression levels of c-Fos in primary hypothalamic neurons detected by qPCR. Experimental treatments were conducted as in Fig. 4F (n = 3 per group). **** p < 0.0001 (one-way ANOVA). F Representative confocal images (scale bar = 10 μm) of c-Fos and CRH in primary hypothalamic neurons overexpressing GFP/ASIC1-GFP or knockdown of ASIC1a for 48 h, before or 15 min after treatment with HBSS at pH 6.5 with or without 100 nM PcTx1 or 200 nM amiloride. G The bar graph illustrated the relative fluorescence intensity of c-Fos shown in Fig. 5F (n = 3 per group). **** p < 0.0001 (one-way ANOVA). H The bar graph shows the secretion levels of CRH in primary hypothalamic neuron culture supernatants detected by ELISA, after treatment with pH 6.5 HBSS or simultaneous treatment with 80 μM T-5224 for 30 min following 48-h overexpression of ASIC1a (n = 3 per group). * p < 0.05 (one-way ANOVA). I Representative western blot assay showing the protein expression levels of CRH, ASIC1a, and c-Fos in primary hypothalamic neurons. Experimental treatments were conducted as in Fig. 5H. J The bar graph illustrates the grayscale scanning analysis of Fig. 5I, showing the relative expression levels of c-Fos (n = 3 per group). * p < 0.05 (one-way ANOVA). K The bar graph illustrates the grayscale scanning analysis of Fig. 5I, showing the relative expression levels of CRH (n = 3 per group). * p < 0.05 (one-way ANOVA). See also Supplementary Data .

Article Snippet: After blocking (5% skimmed milk), the membranes were incubated with primary antibodies overnight, including that CRH/CRF Polyclonal antibody (Cat#10944-1-AP, Proteintech, Wuhan, China), ASIC1a Polyclonal antibody (Cat#27235-1-AP, Proteintech, Wuhan, China), c-Fos monoclonal antibody (Cat#66590-1-Ig, Proteintech, Wuhan, China), phospho-CaMKII alpha/delta (Thr286) antibody (Cat#AF3493, Affinity Biosciences, Jiangsu, China) and CaMKII α antibody (Cat#WL03453, Wanlei Bio, Shenyang, China), and β-actin antibody (Cat#WL01372, Wanlei Bio, Shenyang, China), and then followed by HRP-conjugated secondary antibodies (1:5000).

Techniques: Fluorescence, Cell Culture, Plasmid Preparation, Western Blot, Expressing, Knockdown, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Structural overview (PDB ID 4FZ0) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labeling, highlighted in red. PcTx1 (teal) binds to the subunit interfaces. ( B ) Representative two-electrode voltage-clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in the absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9). Scale bars are 4 µA (vertical) and 60 s (horizontal). ( C ) Same as in ( B ) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. Scale bars are 4 µA (vertical) and 60 s (horizontal). ( D ) Concentration–response relationship of WT ASIC1a activation and SSD in the absence and presence of 30 nM PcTx1 retrieved form experiments shown in ( B ) and ( C ) (n = 6–18). ( E ) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). Scale bars are 60 s (black horizontal), 10 µA (black vertical), and 10% (red vertical). ( F ) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in ( E ) relative to the fluorescence observed upon PcTx1 application. ( G ) Representative trace of a VCF recording of V80C* equivalent to the ones shown in ( E ). Scale bars are 60s (black horizontal), 10 µA (black vertical), and 10% (red vertical).( H ) Same as in ( F ) but for V80C*F350L. Data in ( D ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 1—source data 1. TEVC data from mASIC1a WT of activation and SSD with and without PcTx1, as shown in . Figure 1—source data 2. VCF data from K105C* and V80C* of different PcTx1 washout protocols, as shown in and .

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer; A16110, Thermo Fisher Scientific).

Techniques: Labeling, Activation Assay, Concentration Assay, Fluorescence

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Representative trace of a VCF recording of K105C* with the current in black and the fluorescence in red to establish steady-state desensitization (SSD) and pH-dependent fluorescence response. Inset shows pH 7.0 application without subsequent pH 5.5 activation. ( B ) SSD and fluorescence response curves for different acid-sensing ion channel 1a (ASIC1a) variants based on recording protocols shown in ( A ). Data are presented as mean ± 95 CI, panel ( B) of K105C* is adapted from . Figure 1—figure supplement 1—source data 1. VCF data from mASIC1a K105C* and K105C*F350L of the pH dependent changes in fluorescence and SSD, as shown in . Figure 1—figure supplement 1—source data 2. VCF data from mASIC1a V80C* and V80C*F350L of the pH dependent changes in fluorescence and SSD, as shown in .

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer; A16110, Thermo Fisher Scientific).

Techniques: Fluorescence, Activation Assay

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Voltage-clamp fluorometry (VCF) trace of K105C* showing the introduction of the ‘Global’ inhibitory binding mode upon application of 300 nM PcTx1 at pH 7.4. During washout and repeated activation, the channel readily returns to a functional apo state (current, black trace) while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace), characteristic for the ‘ECD only ’ state. ( B ) VCF traces highlighting the fluorescence changes associated with application of PcTx1 at pH 8.0 with subsequent application of pH 5.5 (left), pH 7.4 (middle), and pH 8.0 (right). Respective PcTx1 binding modes are indicated below the traces. ( C ) Quantitative comparison of the fluorescence signal 60 s into the pH 7.4 application at the end of the experiments shown in ( B ) normalized to the fluorescence change induced by pH 5.5 application. ( D ) Schematic representation of the different pH-dependent binding modes of PcTx1: A ‘Loose’ closed state at high pH, a ‘Global’ state that exists at neutral/low pH that leads to conformational rearrangements in the extracellular domain (ECD) and the pore (indicated in orange), and an ‘ECD only ’ state in which the conformational rearrangements are only found in the ECD and that exists at neutral/low pH even when PcTx1 is absent in the extracellular solution. Teal background shading in the ‘Loose’ and ‘Global’ indicates the presence of PcTx1 in the extracellular solution (although not mandatory, see text for details). ( E ) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s big dynorphin (BigDyn) (1 µM) application (purple bar), with subsequent washout and activation. BigDyn is reapplied after the ‘ECD only ’ state has been evoked through PcTx1 (300 nM) application, this time resulting in a smaller decrease in the fluorescence signal. ( F ) Quantitative comparison of the fluorescence change induced by a 60 s BigDyn application to the apo (control) and to the PcTx1-induced ‘ECD only ’ state (post PcTx1), normalized to the signal induced by pH 5.5. ( G ) VCF trace of K105C* where 300 nM PcTx1 is applied to the ‘ECD only ’ state. ( H ) Quantitative analysis of the protocol shown in ( G ) comparing the fluorescence change induced by PcTx1 to the apo state at 7.4 (control) with the PcTx1 application to the ‘ECD only ’ state. All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( C ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 2—source data 1. VCF data from mASIC1a K105C* of single and multiple activations during PcTx1 washout, as shown in and . Figure 2—source data 2. VCF data from mASIC1a K105C* of PcTx1 application at pH 8.0 followed by different washout protocols, as seen in . Figure 2—source data 3. VCF data of mASIC1a K105C* of BigDyn and PcTx1 application, as seen in and .

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer; A16110, Thermo Fisher Scientific).

Techniques: Binding Assay, Activation Assay, Functional Assay, Fluorescence, Comparison, Control

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Model of the co-crystal structure of cASIC1a and PcTx1 (teal) binding to the extracellular domain (PDBID 4FZO). Inset shows a closeup of the interaction site at the acidic pocket, including acid-sensing ion channel 1a (ASIC1a) residue F350 (orange). ( B ) Representative two-electrode voltage-clamp (TEVC) trace showing mASIC1a F350L pH activation in the absence (top) and presence (bottom) of 30 nM PcTx1. Scale bars are 4 µA (vertical) and 60 s (horizontal). ( C ) Activation and steady-state desensitization (SSD) curve of F350L mASIC1a without (orange) and with (teal) 30 nM PcTx1 (n = 5–12). ( D ) TEVC traces showing the effect of 1 nM PcTx1 on mASIC1a WT (top) and 100 nM PcTx1 on F350L (bottom) applied at pH 7.4. Scale bars are 4 µA (vertical) and 30 s (horizontal). ( E ) PcTx1 concentration–response curve at pH 7.4 using the protocol shown in ( D ) (WT: n = 9–14, F350L: n = 4–7). ( F ) Representative voltage-clamp fluorometry (VCF) trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.4. ( G ) Left: representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.3. Right: comparison of the fluorescence change upon PcTx1 application and after a 3 min washout between K105C* and K150C*F350L. ( H ) Left: representative trace of a VCF recording of V80C*F350L equivalent to the one shown in ( G ). Right: same analysis as in ( G ) but compared between V80C* and V80C*F350L. Data in ( C ), ( E ), ( G ), and ( H ) are presented as mean ± 95 CI, unpaired Mann–Whitney test, **p<0.005, ****p<0.0001. Figure 3—source data 1. TEVC data from mASIC1a F350L of activation and SSD with and without PcTx1, as shown in . Figure 3—source data 2. TEVC data from mASIC1a WT and F350L of PcTx1 concentration-response curve, as shown in . Figure 3—source data 3. VCF data from mASIC1a K105C*(F350L) and V80C*(F350L) of application and washout of PcTx1, as shown in .

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer; A16110, Thermo Fisher Scientific).

Techniques: Binding Assay, Residue, Activation Assay, Concentration Assay, Mutagenesis, Comparison, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: ( A ) Schematic representation of the concatemer design showing mASIC1a sequence in teal, linkers that connect subunits a, b, and c in red, with restriction sites underlined. ( B ) Primer pairs used to generate inserts a–c for ligation into the concatemer constructs. Nucleotide spacers and untranslated sequences are shown in gray, restriction sites are shown in red, peptide linker are shown in black, and mASIC1a sequence is in teal. ( C ) Western blot of surface-purified membrane protein from oocytes expressing monomeric or concatemeric acid-sensing ion channel 1a (ASIC1a) constructs using a mASIC1a-specific antibody. Bands for monomeric ASIC1a WT and F350L subunits appear at ~60 kDa while the bands for concatemeric ASIC1a constructs appear at ~180 kDa, corresponding to three covalently linked ASIC1a subunits. Panels ( A, B ) adapted from .

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer; A16110, Thermo Fisher Scientific).

Techniques: Sequencing, Ligation, Construct, Western Blot, Purification, Membrane, Expressing

Journal: eLife

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.7554/eLife.73384

Figure Lengend Snippet: Schematic representation of a side view of acid-sensing ion channel 1a (ASIC1a) extracellular domain (ECD) and transmembrane domain (TMD) and top view of the three subunits and consequences of PcTx1 (teal) binding at neutral/low pH (as in ) and with the F350L mutation (orange) in 0–3 subunits. The side view coloring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ state with increasing number of F350L-containing subunits, and the decreasing inhibitory effect on the pore. In channels with a single F350L subunit, only the PcTx1-induced conformational state of the ECD is affected, while the TMD behaves WT-like.

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer; A16110, Thermo Fisher Scientific).

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.1101/2021.06.21.449215

Figure Lengend Snippet: (A) Structural overview (PDB ID 4FZO) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labelling, highlighted in red. PcTx1 (teal) binds to the subunit interfaces. (B) Representative two-electrode voltage clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9) (C) Same as in (B) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. (D) Concentration-response relationship of WT ASIC1a activation and SSD in absence and presence of 30 nM PcTx1 retrieved form experiments shown in (B) and (C). (E) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). (F) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in B relative to the fluorescence observed upon PcTx1 application. (G) Representative trace of a VCF recording of V80C* equivalent to the ones shown in (E). (H) Same as in (F) but for V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B-C) and 10 μA (E and G) (black vertical), and 10% (red, E and G only). Data in D, F and H are presented as mean + 95CI; n = 3-18 for individual data points in D.

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer) (A16110, Thermo Fisher Scientific).

Techniques: Activation Assay, Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.1101/2021.06.21.449215

Figure Lengend Snippet: (A) VCF trace of K105C* showing that the channel readily returns to a functional ‘apo’ state (current, black trace) after application of 300 nM PcTx1 at pH 7.4, while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace). (B) VCF traces highlighting the fluorescence changes associated with application of PcTx1 at pH 8.0 with subsequent application of pH 5.5 (left), pH 7.4 (middle) and pH 8.0 (right). (C) Quantitative comparison of the fluorescence signal 60 s into the pH 7.4 application at the end of the experiments shown in (B) normalized to the fluorescence change induced by pH 5.5 application. (D) Schematic representation of the different pH dependent binding modes of PcTx1: A ‘Loose’ closed state at high pH, a ‘Global’ state that exists at neutral/low pH in the presence of PcTx1 and leads to conformational rearrangements in both ASIC1a ECD and pore (indicated in orange), and an ‘ECD only ’ state in which the conformational rearrangements are only found in the ECD and that exists at neutral/low pH even when PxTx1 is absent in the extracellular solution. Teal background shading indicates the presence of PcTx1 in the extracellular solution. Transitions between the binding modes that are explicitly shown in this work are indicated in full opacity. (E) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s BigDyn (1 μM) application (purple bar), with subsequent washout and activation. BigDyn is then applied again after the ‘ECD only ’ state is evoked through PcTx1 (300 nM) application, this time only resulting in a slow decrease in the fluorescence signal. (F) Quantitative comparison of the fluorescence change induced by a 60 s BigDyn application to the ‘apo’ (Control) and to the PcTx1-induced ‘ECD only ’ state (Post PcTx1), normalized to the signal induced by pH 5.5; and of the fluorescence signal induced by PcTx1 at pH 7.4 (Control) and BigDyn pre-application (Post BigDyn), respectively. Scale bars are 60 s (black horizontal), 10 μA (black vertical), and 10% (A and E) or 5 % (B) (red, A, B and E only). Data in C and F are presented as mean + 95CI.

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer) (A16110, Thermo Fisher Scientific).

Techniques: Functional Assay, Fluorescence, Binding Assay, Activation Assay

Journal: bioRxiv

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.1101/2021.06.21.449215

Figure Lengend Snippet: (A) Model of the co-crystal structure of cASIC1a and PcTx1 (teal) binding to the extracellular domain (PDBID 4FZO). Inset shows a close up of the interaction site at the acidic pocket, including ASIC1a residue F350 (orange). (B) Representative TEVC trace showing mASIC1a F350L pH activation in the absence (top) and presence (bottom) of 30 nM PcTx1. (C) Activation and SSD curve of F350L mASIC1a without (orange) and with (teal) PcTx1 (n=5–12). (D) TEVC traces showing the effect of 1 nM PcTx1 on mASIC1a WT (top) and 100 nM PcTx1 on F350L (bottom) applied at pH 7.4. (E) PcTx1 concentration-response curve at pH 7.4 using the protocol shown in (D) (n=4–14). (F) Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.4. (G) Left: Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.3. Right: Comparison of the fluorescence change upon PcTx1 application and after a 3 min washout between K105C* and K150C*F350L (H) Left: Representative trace of a VCF recording of V80C*F350L equivalent to the one shown in G. Right: Same analysis as in G but compared between V80C* and V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B and D only) and 10 μA (black vertical), and 10% (red) (F-H). Data in C, E, G and H are presented as mean + 95CI.

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer) (A16110, Thermo Fisher Scientific).

Techniques: Binding Assay, Activation Assay, Concentration Assay, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

doi: 10.1101/2021.06.21.449215

Figure Lengend Snippet: Schematic representation of a sideview of ASIC1a ECD and TMD and top view of the three subunits and consequences of PcTx1 (teal) binding at neutral/low pH (as in ) and with the F350L mutation (orange) in 0–3 subunits. The side view colouring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ conformation with increasing number of F350L containing subunits, and the decreasing inhibitory effect on the pore. In channels with a single F350L subunit, only the PcTx1-induced conformational state of the ECD is affected, while the TMD behaves WT-like.

Article Snippet: The blot was then incubated for 10 min at room temperature with a 1:1000 dilution of rabbit polyclonal anti-ASIC1a antibody (OSR00097W, Thermo Fisher Scientific) in blocking buffer followed by 10 min incubation at room temperature with goat anti-rabbit IgG/horseradish peroxidase-conjugate (1:10,000 dilution of 1 mg/mL stock in blocking buffer) (A16110, Thermo Fisher Scientific).

Techniques: Binding Assay, Mutagenesis

Journal: Journal of Inflammation Research

Article Title: Estrogen Protects Articular Cartilage by Downregulating ASIC1a in Rheumatoid Arthritis

doi: 10.2147/JIR.S295222

Figure Lengend Snippet: Estradiol reduces cartilage damage and protects articular cartilage in ovariectomized AA rats in vivo. ( A ) Normal and AA rat right hind ankle joint sections stained with toluidine blue staining. ( B ) Normal and AA rat right hind ankle joint sections stained with HE staining. ( C ) The scores of cartilage damage (n = 7), Semiquantitative analysis of ASIC1a protein expression in ankle joint of the E2 group and the AA group (n = 7) were calculated using the IPP 6.0 software. ( D ) Representative image of paw edema in rats and the joint swelling of the AA group and the normal group were measured by the joint swelling instrument (n = 7). ( E ) The body weight of all groups (n = 7). ( F ) Immunohistochemical of ASIC1a in ankle joint of the AA group and the E2 group (n = 7). ( G ) Semiquantitative analysis of ASIC1a protein expression in ankle joint of the E2 group and the AA group (n = 7) were calculated using the IPP 6.0 software ( H ) Western blot analysis of ASIC1a expressed in ankle joint of the E2 group and the AA group (n = 7). β-actin served as a loading normal, and the indicated proteins were quantified with Image J software. ( I ) Representative image of paw edema in rats with adjuvant-induced arthritis (n = 7). ( J ) The joint swelling of all groups was measured by the joint swelling instrument (n = 7). ( K ) HE staining in ankle joint of all groups (n = 7). ( L ) Toluidine blue staining in ankle joint of all groups (n = 7). ( M ) The scores of cartilage damage. Data are presented as the mean ± SEM of three independent experiments, ( C ) ***P < 0.001 compared with the Normal group. ( G and H ) **P < 0.01, ***P < 0.001 versus the AA group. ( M ) ***P < 0.001 compared with the solvent-treated group.

Article Snippet: Rabbit anti-ASIC1a antibody was purchased from Bioss (Beijing, China) (bs-2586R).

Techniques: In Vivo, Staining, Expressing, Software, Immunohistochemical staining, Western Blot

Journal: Journal of Inflammation Research

Article Title: Estrogen Protects Articular Cartilage by Downregulating ASIC1a in Rheumatoid Arthritis

doi: 10.2147/JIR.S295222

Figure Lengend Snippet: Estradiol-mediated protection against the autophagy of articular cartilage in OVX rats with AA in vivo. ( A ) The immunohistochemistry results of GPER1 and ERα of the Normal group and the AA+E2 group. Semiquantitative analysis of GPER1 and ERα expression in ankle joint of each group (n = 7) were calculated using the IPP 6.0 software. ( B ) Immunohistochemistry analysis of Beclin1, LC3, p62 and ASIC1a in articular cartilage tissue of all groups (n = 7). ( C ) Semi-quantitative analysis of ASIC1a, Beclin1, LC3, p62 expression in rat cartilage by integral optical density (IOD). ( D ) The protein expression levels of Beclin1, LC3, ASIC1a and p62 in articular cartilage tissue of all groups were measured by Western blot. β-actin served as a loading normal, and the indicated proteins were quantified with Image J software. ( E ) Estradiol, IL-1β and TNF-α levels in the serum of all groups were measured by ELISA assay (n = 7). Data are presented as the mean ± SEM of three independent experiments. ( A ) ## P < 0.01, ### P < 0.001 compared with the Normal group. ( C – E ) *P < 0.05, **P < 0.01, ***P < 0.001 compared with the solvent-treated group.

Article Snippet: Rabbit anti-ASIC1a antibody was purchased from Bioss (Beijing, China) (bs-2586R).

Techniques: In Vivo, Immunohistochemistry, Expressing, Software, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: Estrogen Protects Articular Cartilage by Downregulating ASIC1a in Rheumatoid Arthritis

doi: 10.2147/JIR.S295222

Figure Lengend Snippet: Estradiol contributes to autophagy in chondrocytes by mediating the downregulation of ASIC1a. ( A and B ) The expression of ASIC1a decreased in a time- and concentration-dependent manner following stimulation with 0–2000 nmol/mL estradiol for 0 h to 48 h in chondrocytes. ( C and D ) The expression of Beclin1, LC3 and p62 protein in chondrocytes in a time- and concentration-dependent manner following stimulation with 0–2000 nmol/mL estradiol for 0 h to 48 h. ( E ) Estradiol induced expression levels of ASIC1a in chondrocytes cultured in pH 6.0 medium for 24 h with or without E2 (500 nmol/mL) were analyzed by immunofluorescence. Representative views from each group are presented (original magnification, ×400). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus 0 nmol/mL group; # P < 0.05, ## P < 0.01 versus 0 h group.

Article Snippet: Rabbit anti-ASIC1a antibody was purchased from Bioss (Beijing, China) (bs-2586R).

Techniques: Expressing, Concentration Assay, Cell Culture, Immunofluorescence

Journal: Journal of Inflammation Research

Article Title: Estrogen Protects Articular Cartilage by Downregulating ASIC1a in Rheumatoid Arthritis

doi: 10.2147/JIR.S295222

Figure Lengend Snippet: Estradiol modulates ASIC1a through its receptor GPER1.( A ) Western blot analysis of the protein expression of ASIC1a with or without for estradiol (500 nmol/mL), MPP (20 mmol/L), and G15 (15 μmol/L). ( B ) Western blot analysis of ERα and GPER1 protein expressions in chondrocytes that without transfection or transfected with negative control (NC) or siRNA for ERα or GPER1. ( C ) Western blot analysis of the protein expression of ASIC1a with or without for estradiol (500 nmol/mL) and ERα-siRNA or GPER1-siRNA. ( D – F ) Chondrocytes in serum-free confluent monolayer cultures were stimulated by with increasing concentrations of G1 (0–1000 nmol/L) and levels of ASIC1a, GPER1, Beclin1, LC3 and p62 proteins were measured by Western blot. Data are presented as mean ± SEM, *P < 0.05, versus pH + E2 group, ^ P < 0.05, ^^ P < 0.01 versus the NC group, $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 versus 0 nmol/L.

Article Snippet: Rabbit anti-ASIC1a antibody was purchased from Bioss (Beijing, China) (bs-2586R).

Techniques: Western Blot, Expressing, Transfection, Negative Control

Journal: Journal of Inflammation Research

Article Title: Estrogen Protects Articular Cartilage by Downregulating ASIC1a in Rheumatoid Arthritis

doi: 10.2147/JIR.S295222

Figure Lengend Snippet: GPER1 inhibits autophagy by down-regulating the expression of ASIC1a. ( A – C ) Western blot analysis of ASIC1a, GPER1, Beclin1, LC3 and p62 protein in chondrocytes were pretreated with or without agonist for GPER1 (G1) followed by stimulation with G15. ( D and E ) The mRNA expression of ASIC1a, Beclin1, LC3 and Atg5 in chondrocytes were pretreated with or without G1 followed by stimulation with G15. ( F – I ) G1 and G15-induced expression levels of ASIC1a, GPER1, Beclin1 and LC3 were analyzed by immunofluorescence in chondrocytes. Representative views from each group are presented (original magnification, ×400). Data are presented as mean ± SEM. ***P < 0.001 versus normal group; ## P < 0.01, ### P < 0.001 versus pH group; $ P < 0.05, $$ P < 0.01 versus G1 group.

Article Snippet: Rabbit anti-ASIC1a antibody was purchased from Bioss (Beijing, China) (bs-2586R).

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Journal of Inflammation Research

Article Title: Estrogen Protects Articular Cartilage by Downregulating ASIC1a in Rheumatoid Arthritis

doi: 10.2147/JIR.S295222

Figure Lengend Snippet: GPER1 inhibits the expression of ASIC1a by activating the PI3K-AKT-mTOR signaling in chondrocytes. ( A ) Cultured chondrocytes were pretreated with or without agonist for GPER1 (G1) followed by stimulation with G15 and levels of phosphorylated and total mTOR, AKT and S6K1 proteins were measured by Western blot. ( B ) Cultured chondrocytes were pretreated with or without inhibitors for AKT (MK2206, 1000 nmol/mL) followed by stimulation with G1 and levels of phosphorylated and total mTOR, AKT and S6K1 proteins were measured by Western blot. ( C and D ) qRT-PCR analysis of ASIC1a, Beclin1, LC3 and Atg5 mRNA expression levels in chondrocytes. ( E ) Western blot analysis of Beclin1, LC3, p62 and ASIC1a protein expressions in chondrocytes that without transfection or transfected with negative control (NC), ASIC1a-RNAi or PCTX-1 (100 nmol/L). ( F ) qRT-PCR analysis of Beclin1, LC3 and Atg5 protein expressions in chondrocytes that without transfection or transfected with negative control (NC), ASIC1a-RNAi or PCTX-1 (100 nmol/L). Data are presented as mean ± SEM. ( A – D ) *P < 0.05, **P < 0.01 versus normal group; # P < 0.05, ## P < 0.01, ### P < 0.001 versus pH group; $ P < 0.05, $$ P < 0.01 versus G1 group. ( E and F ) *P < 0.05, ***P < 0.001 versus normal group; # P < 0.05, ## P < 0.01, ### P < 0.001 versus pH group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 versus NC group.

Article Snippet: Rabbit anti-ASIC1a antibody was purchased from Bioss (Beijing, China) (bs-2586R).

Techniques: Expressing, Cell Culture, Western Blot, Quantitative RT-PCR, Transfection, Negative Control