rabbit anti-asic2  (Alpha Diagnostics)

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Primers and bp product size used for genotyping, RT-PCR, and qPCR for ASIC1, ASIC2, ASIC3, β-actin, and RNA18S

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques: Sequencing

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Acid-sensing ion channel (ASIC)2 and ASIC3 are expressed in pulmonary arterial smooth muscle cells (PASMC). Representative electrophoresis gel showing reverse transcriptase (RT)-PCR for ASIC2 (240 bp), ASIC3 (432 bp), and β-actin (294 bp) in PASMC from wild-type (WT), ASIC2−/−, and ASIC3−/− mice.

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques: Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Average systemic MABP (mm Hg) and HR in beats/min in conscious WT, ASIC2 −/− , and ASIC3 −/− male mice by radiotelemetry recorded over 72 h

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques:

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Chronic hypoxia (CH) does not significantly alter acid-sensing ion channel (ASIC)2 or ASIC3 protein levels in intrapulmonary arteries. A: representative Western blots of ASIC2 (predicted ∼69 kDa), ASIC3 (predicted ∼59 kDa), and corresponding Coomassie-stained blot. All samples were collected at the same time and processed in parallel on 2 gels for each antibody to accommodate 12 samples [6 control (Con) and 6 CH] from male wild-type (WT) mice. B: summary data detecting ASIC2/3 expression (normalized to entire lane on Coomassie-stained blot) in isolated intrapulmonary arteries from Con and CH WT mice. Values are means ± SE; dots indicate n = 6/group, analyzed by unpaired t-test.

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques: Western Blot, Staining, Expressing, Isolation

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Right ventricular systolic pressure (RVSP) is augmented in ASIC2−/− mice. Summary data for RVSP (A) and ratio of right ventricular (RV) to left ventricular plus septum (LV + S) heart weight (B) in anesthetized wild-type (WT), acid-sensing ion channel (ASIC)2−/−, and ASIC3−/− mice following exposure control or chronic hypoxia (CH) conditions. Values are means ± SE; n/group are indicated at the bottom of the bars as no. of males/females and as color-coded dots: black circle, control male; red circle, control female; black square, CH male; red square, CH female. There is no significant difference between males and females (P = 0.62). ****P < 0.0001 vs. respective control (Con) and ####P < 0.0001 vs. corresponding WT, analyzed by 2-way ANOVA and individual groups compared using Tukey’s multiple-comparisons test.

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques:

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Mean BW (g), RVDP (mmHg), HR in beats/min, RV CI (1/s), and HCT (%red blood cells) in WT, ASIC2 −/− , and ASIC3 −/− mice exposed to Con or CH conditions

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques:

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Small pulmonary arterial muscularization is greater in acid-sensing ion channel (ASIC)2−/− compared with wild-type (WT) mice. A: representative smooth muscle α-actin immunofluorescence images of lung sections from control (Con) and chronic hypoxia (CH) WT, ASIC2−/−, and ASIC3−/− mice. Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. B: %muscularization calculated as %thresholded smooth muscle α-actin area divided by total arterial wall area. All vessels were <100 µm. Values are means ± SE; n = no. of vessels (indicated in bar) analyzed from 4 animals/group. ****P ≤ 0.0001 vs. the control group; ####P < 0.0001 vs. corresponding WT mice.

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques: Immunofluorescence, Fluorescence

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Effect of acid-sensing ion channel (ASIC) on baseline ventilation and blood gases following chronic hypoxia (CH). Whole body plethysmography was used to determine respiratory frequency (breaths/min; A), tidal volume (μl·breath−1·g body wt−1; B), and minute ventilation (ml·min−1·g body wt−1; C) in conscious wild-type (WT), ASIC2−/−, and ASIC3−/− CH mice breathing room air. Baseline arterial Po2 (mmHg; D), Pco2 (mmHg; E), and pH (F) in conscious WT, ASIC2−/−, and ASIC3−/− CH mice breathing room air. Values are means ± SE; n/group are indicated at the bottom of the bars as no. of males/females and as color-coded dots: black square, CH male; red square, CH female. There is no significant difference between males and females (P = 0.979). *P < 0.05 and **P < 0.01 vs. respective WT, analyzed by 1-way ANOVA and individual groups compared using Tukey’s multiple-comparisons test.

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques:

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Blood gas measurements and ventilatory responses in response to hypoxia, isocapnic hypoxia, or hypercapnia in conscious, chronically catheterized WT, ASIC2 −/− , and ASIC3 −/− mice following 4 wk of CH

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques:

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Enhanced pulmonary vasoconstrictor reactivity in control acid-sensing ion channel (ASIC)2−/− and ASIC3−/− mice. Baseline pulmonary vascular resistance (PVR; in mmHg·ml−1·min·kg−1; A) and changes (Δ) in PVR in response to hypoxic ventilation (6% CO2 and balance N2; B), KCl (30 mM; C), and U-46619 (10−9 – 10−6 M; D) in isolated lungs from control wild-type (WT), ASIC2−/−, and ASIC3−/− mice. All experiments were conducted in the presence of NG-nitro-l-arginine (300 μM) and meclofenamate (30 μM). Values are means ± SE; n/group indicated as color-coded dots: black circle, control male; red circle, control female. **P < 0.01 and ***P < 0.001 vs. corresponding WT, analyzed by 1-way ANOVA and individual groups compared using Tukey’s multiple comparisons test. NS, not significant.

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques: Isolation

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Augmented control acid-sensing ion channel (ASIC)1-dependent basal Ca2+ and store-operated calcium entry (SOCE) in pulmonary arterial smooth muscle cells (PASMC) from ASIC2−/− mice. A: summary data showing basal and Ca2+ free [Ca2+]i levels (fura2 340/380 ratio) in PASMC from control wild-type (WT), ASIC2−/−, or ASIC3−/− male mice. B: [Ca2+]i levels (fura2 340/380 ratio) following treatment with diltiazem (50 μM) or PcTX1 (20 nM). C: SOCE responses performed in the presence of cyclopiazonic acid (CPA; 10 μM), diltiazem (50 μM), and ± PcTX1. Values are means ± SE; ●, n of 6–10/group. *P < 0.05 and ****P < 0.0001 vs. WT; #P < 0.05 and ####P < 0.0001 vs. corresponding baseline/vehicle, analyzed by 2-way ANOVA and individual groups compared using Tukey’s multiple comparisons test.

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques:

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Enhanced pulmonary vasoconstrictor reactivity in control acid-sensing ion channel (ASIC)2−/− mice is mediated by ASIC1. A and B: basal arterial tone (%Ca2+ free diameter; A) and basal intracellular Ca2+ concentration ([Ca2+]i; B) expressed as vessel wall fura-2 ratio (F340/F380) in isolated, pressurized, small pulmonary arteries from control wild-type (WT) and ASIC2−/− mice in the presence or absence of psalmotoxin 1 (PcTX1; 20 nM). C and D: %vasoconstriction (C) and changes (Δ) in vessel wall Ca2+ (Δ in fura-2 ratio) (D) in response to KCl (30 mM). E–H: %vasoconstriction (E) and change in vessel wall Ca2+ (G) to U-46619 (10−10 to 10−6 M). U-46619 dose response curves were analyzed by evaluating maximum effect (Emax; E and G) and logEC50 (F and H). Values are means ± SE; ●, n/group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. corresponding WT; #P < 0.05, ##P < 0.01, and ###P < 0.001, and ####P < 0.0001 vs. corresponding vehicle (Veh) treated, analyzed by 2-way ANOVA and individual groups compared using Tukey’s multiple comparisons test.

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

Techniques: Concentration Assay, Isolation

Journal: Journal of Applied Physiology

Article Title: Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension

doi: 10.1152/japplphysiol.00894.2018

Figure Lengend Snippet: Effect of acid-sensing ion channel (ASIC)2−/− and ASIC3−/− on ASIC1 expression. A: summary data showing mRNA analysis by real-time quantitative PCR for ASIC1 in primary pulmonary arterial smooth muscle cells (PASMC) from control wild-type (WT), ASIC2−/−, and ASIC3−/− mice. ASIC1 expression was normalized to 18S rRNA as an internal control (2–ΔΔCT). B and C: representative Western blots for ASIC1 (indicated by arrow; B) and summary data for protein analysis of ASIC1 (normalized to entire lane on Coomassie-stained blot; C) in primary PASMC from WT, ASIC2−/−, and ASIC3−/− mice. Values are means ± SE. Nos. of animals/group (6, 7) are indicated as color-coded dots: black circle, control male; red circle, control female, analyzed by 1-way ANOVA.

Article Snippet: Blots were blocked for 1 h with 5% milk then incubated for 48 h at 4°C with rabbit anti-ASIC1 (1:500, no. ab5674P; Millipore), rabbit anti-ASIC2 (1:500, no. ab5460; Millipore), or rabbit anti-ASIC3 (1:400, no. ab49333; Abcam).

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