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Santa Cruz Biotechnology goat anti asic1
Effect of 30c and Let-7 on ASIC1a expression. CHO cells were transfected with ASIC1a-UTR together with the vector control, or expression constructs for miR-30c or let-7. Two days after transfection, cells were lysed and analyzed by Western blot using an <t>anti-ASIC1</t> and an anti-b-tubulin antibodies. Representative blots and quantification of ASIC1a: tubulin ratio was shown. Asterisk indicates significant (p
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Images

1) Product Images from "miR-149 reduces while let-7 elevates ASIC1a expression in vitro"

Article Title: miR-149 reduces while let-7 elevates ASIC1a expression in vitro

Journal: International Journal of Physiology, Pathophysiology and Pharmacology

doi:

Effect of 30c and Let-7 on ASIC1a expression. CHO cells were transfected with ASIC1a-UTR together with the vector control, or expression constructs for miR-30c or let-7. Two days after transfection, cells were lysed and analyzed by Western blot using an anti-ASIC1 and an anti-b-tubulin antibodies. Representative blots and quantification of ASIC1a: tubulin ratio was shown. Asterisk indicates significant (p
Figure Legend Snippet: Effect of 30c and Let-7 on ASIC1a expression. CHO cells were transfected with ASIC1a-UTR together with the vector control, or expression constructs for miR-30c or let-7. Two days after transfection, cells were lysed and analyzed by Western blot using an anti-ASIC1 and an anti-b-tubulin antibodies. Representative blots and quantification of ASIC1a: tubulin ratio was shown. Asterisk indicates significant (p

Techniques Used: Expressing, Transfection, Plasmid Preparation, Construct, Western Blot

2) Product Images from "Human ASIC1a mediates stronger acid-induced responses as compared with mouse ASIC1a"

Article Title: Human ASIC1a mediates stronger acid-induced responses as compared with mouse ASIC1a

Journal: The FASEB Journal

doi: 10.1096/fj.201701367R

hASIC1a and mASIC1a S285P elicit a larger acid-activated calcium increase. CHO cells were transfected with various ASIC1 constructs, as indicated together with cameleon YC3.60, a ratiometric calcium reporter. FRET imaging and spectral unmixing were performed, as described in Materials and Methods. A ) Representative images showing the fluorescent images of transfected cells (leftmost) and ratiometric images (right 2 sets) before, at peak, and after stimulation with a pH 6.5 solution. For ratio images, red and blue indicate high and low FRET, respectively. B ) Average time-dependent changes in FRET (YFP/CFP ratio) of cells expressing mASIC1a, hASIC1a, and mASIC1a S285P. C ) Quantification of peak change in FRET (ΔFRET) of cells expressing the 3 different constructs. Bar numbers indicate total number of cells analyzed ( B, C ). Asterisk indicates differences from mASIC1a (ANOVA). Numbers in bars indicate number of repeats.
Figure Legend Snippet: hASIC1a and mASIC1a S285P elicit a larger acid-activated calcium increase. CHO cells were transfected with various ASIC1 constructs, as indicated together with cameleon YC3.60, a ratiometric calcium reporter. FRET imaging and spectral unmixing were performed, as described in Materials and Methods. A ) Representative images showing the fluorescent images of transfected cells (leftmost) and ratiometric images (right 2 sets) before, at peak, and after stimulation with a pH 6.5 solution. For ratio images, red and blue indicate high and low FRET, respectively. B ) Average time-dependent changes in FRET (YFP/CFP ratio) of cells expressing mASIC1a, hASIC1a, and mASIC1a S285P. C ) Quantification of peak change in FRET (ΔFRET) of cells expressing the 3 different constructs. Bar numbers indicate total number of cells analyzed ( B, C ). Asterisk indicates differences from mASIC1a (ANOVA). Numbers in bars indicate number of repeats.

Techniques Used: Transfection, Construct, Imaging, Expressing

Expression of ASIC1 and -2 in human cortical tissue. A ) A typical set of images and traces showing the identification of epileptic foci in 1 patient. Diagram illustrates the layout of the recording electrodes (top left), whereas the MRIs on the right illustrate the location of the epileptic foci (indicated by a red dot) in hippocampus. EEG traces from neocortex and hippocampus: hippocampus exhibited epileptic patterns of activities, which were absent in cortical traces (bottom). Note that the cortical samples were used in this study for comparing ASIC expression between human and mouse brain. B ). Human and mouse tissues were not statistically different in the relative ratio of ASIC1a:ASIC2a ( P > 0.05, either by Student’s t test or Wilcoxon signed rank test) (middle). The relative ASIC2b:ASIC2a ratio was significantly lower in human tissue. P = 0.006 (Student’s t test) (right). C ) Representative Western blot images and quantification showing ASIC1a levels in membrane (left). Membrane fractions from human and mouse cortical tissues were prepared, as described in Materials and Methods. Total lysate, membrane fraction, and cytosol fraction were blotted for ASIC1a, N-cadherin, and β-tubulin III. The latter 2 proteins served as controls for the quality of the preparation. Right: quantification of relative membrane:total ASIC1a ratios. Asterisks ( B , C ) indicate significant differences between mouse and human. Numbers in bars indicate number of repeats.
Figure Legend Snippet: Expression of ASIC1 and -2 in human cortical tissue. A ) A typical set of images and traces showing the identification of epileptic foci in 1 patient. Diagram illustrates the layout of the recording electrodes (top left), whereas the MRIs on the right illustrate the location of the epileptic foci (indicated by a red dot) in hippocampus. EEG traces from neocortex and hippocampus: hippocampus exhibited epileptic patterns of activities, which were absent in cortical traces (bottom). Note that the cortical samples were used in this study for comparing ASIC expression between human and mouse brain. B ). Human and mouse tissues were not statistically different in the relative ratio of ASIC1a:ASIC2a ( P > 0.05, either by Student’s t test or Wilcoxon signed rank test) (middle). The relative ASIC2b:ASIC2a ratio was significantly lower in human tissue. P = 0.006 (Student’s t test) (right). C ) Representative Western blot images and quantification showing ASIC1a levels in membrane (left). Membrane fractions from human and mouse cortical tissues were prepared, as described in Materials and Methods. Total lysate, membrane fraction, and cytosol fraction were blotted for ASIC1a, N-cadherin, and β-tubulin III. The latter 2 proteins served as controls for the quality of the preparation. Right: quantification of relative membrane:total ASIC1a ratios. Asterisks ( B , C ) indicate significant differences between mouse and human. Numbers in bars indicate number of repeats.

Techniques Used: Expressing, Western Blot

3) Product Images from "Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension"

Article Title: Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00269.2013

CH does not alter ASIC1 protein levels in small pulmonary arteries. A : representative Western blots of ASIC1 and GAPDH (20 μg protein/lane). B : summary data for Western blot analysis of ASIC1/GAPDH protein expression. Values are means ± SE; n values are indicated in bars.
Figure Legend Snippet: CH does not alter ASIC1 protein levels in small pulmonary arteries. A : representative Western blots of ASIC1 and GAPDH (20 μg protein/lane). B : summary data for Western blot analysis of ASIC1/GAPDH protein expression. Values are means ± SE; n values are indicated in bars.

Techniques Used: Western Blot, Expressing

ASIC1 protein expression in small pulmonary arteries. Smooth muscle α-actin (SM α-actin) and ASIC1 immunofluorescence in ASIC1 +/+ and ASIC1 −/− lung sections demonstrate the presence of ASIC1 in small pulmonary arteries (∼70–100 μm). Fluorescence images were digitally inverted using ImageJ to provide better contrast and visibility of immunofluorescence. The bottom images represent digitally zoomed fluorescence overlay images corresponding to the boxes in the images of smooth muscle α-actin (green; top ) and ASIC1 (red; middle ).
Figure Legend Snippet: ASIC1 protein expression in small pulmonary arteries. Smooth muscle α-actin (SM α-actin) and ASIC1 immunofluorescence in ASIC1 +/+ and ASIC1 −/− lung sections demonstrate the presence of ASIC1 in small pulmonary arteries (∼70–100 μm). Fluorescence images were digitally inverted using ImageJ to provide better contrast and visibility of immunofluorescence. The bottom images represent digitally zoomed fluorescence overlay images corresponding to the boxes in the images of smooth muscle α-actin (green; top ) and ASIC1 (red; middle ).

Techniques Used: Expressing, Immunofluorescence, Fluorescence

ASIC1 contributes to small pulmonary artery remodeling after CH. A : representative bright-field ( left ) or smooth muscle α-actin immunofluorescence ( right ) images of lung sections from control and CH ASIC1 +/+ and ASIC1 −/− mice. Bottom images show higher-magnification images of ∼10-μm arteries from CH ASIC1 +/+ and ASIC1 −/− mice. The arrow bottom middle image indicates an alveolar duct smooth muscle cell. Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. B –60 μm) from 20 random images/lung section. C : percent muscularization calculated as percent thresholded smooth muscle α-actin area divided by total arterial wall area. Values are means ± SE; n = 4 animals/group. * P ≤ 0.05 vs. the control group; # P
Figure Legend Snippet: ASIC1 contributes to small pulmonary artery remodeling after CH. A : representative bright-field ( left ) or smooth muscle α-actin immunofluorescence ( right ) images of lung sections from control and CH ASIC1 +/+ and ASIC1 −/− mice. Bottom images show higher-magnification images of ∼10-μm arteries from CH ASIC1 +/+ and ASIC1 −/− mice. The arrow bottom middle image indicates an alveolar duct smooth muscle cell. Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. B –60 μm) from 20 random images/lung section. C : percent muscularization calculated as percent thresholded smooth muscle α-actin area divided by total arterial wall area. Values are means ± SE; n = 4 animals/group. * P ≤ 0.05 vs. the control group; # P

Techniques Used: Immunofluorescence, Mouse Assay, Fluorescence

Restoration of acid-sensing ion channel 1 (ASIC1) in pulmonary arterial smooth muscle cells (PASMCs) from ASIC1 knockout (ASIC1 −/− ) mice rescues store-operated Ca 2+ entry (SOCE). A : RT-PCR for ASIC1 and β-actin in PASMCs from ASIC1 +/+ and ASIC1 −/− mice transfected with human (h)ASIC1. B : smooth muscle-22α (SM22α; green) and ASIC1 (red) immunofluorescence in PASMCs from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. C : summary data showing SOCE-induced changes (Δ) in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [expressed as changes in the 340-to-380-nm fluorescence ratio (ΔF 340 /F 380 )] in PASMCs isolated from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. All experiments were performed in the presence of cyclopiazonic acid (CPA; 10 μM) and diltiazem (50 μM). Values are means ± SE; numbers of animals ( n values) are indicated in bars. * P
Figure Legend Snippet: Restoration of acid-sensing ion channel 1 (ASIC1) in pulmonary arterial smooth muscle cells (PASMCs) from ASIC1 knockout (ASIC1 −/− ) mice rescues store-operated Ca 2+ entry (SOCE). A : RT-PCR for ASIC1 and β-actin in PASMCs from ASIC1 +/+ and ASIC1 −/− mice transfected with human (h)ASIC1. B : smooth muscle-22α (SM22α; green) and ASIC1 (red) immunofluorescence in PASMCs from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. C : summary data showing SOCE-induced changes (Δ) in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [expressed as changes in the 340-to-380-nm fluorescence ratio (ΔF 340 /F 380 )] in PASMCs isolated from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. All experiments were performed in the presence of cyclopiazonic acid (CPA; 10 μM) and diltiazem (50 μM). Values are means ± SE; numbers of animals ( n values) are indicated in bars. * P

Techniques Used: Knock-Out, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunofluorescence, Concentration Assay, Fluorescence, Isolation

ASIC1 contributes to enhanced receptor-mediated vasoconstriction after CH in small pulmonary arteries. A–F : vasoconstriction (percent baseline diameter; A , C , and E ) and changes in arterial wall [Ca 2+ ] i (ΔF 340 /F 380 ; B , D , and F ) to UTP (10 −7 to 3 × 10 −4 M; A and B ), endothelin-1 (ET-1; 10 −11 to 10 −7 M; C and D ), and depolarizing concentrations of KCl (10 −1.75 to 10 −1.00 M; E and F ) in small pulmonary arteries from control and CH ASIC1 +/+ and ASIC1 −/− mice. Values are means ± SE; n = 5–7 animals/group. * P ≤ 0.05 vs. the control group; # P
Figure Legend Snippet: ASIC1 contributes to enhanced receptor-mediated vasoconstriction after CH in small pulmonary arteries. A–F : vasoconstriction (percent baseline diameter; A , C , and E ) and changes in arterial wall [Ca 2+ ] i (ΔF 340 /F 380 ; B , D , and F ) to UTP (10 −7 to 3 × 10 −4 M; A and B ), endothelin-1 (ET-1; 10 −11 to 10 −7 M; C and D ), and depolarizing concentrations of KCl (10 −1.75 to 10 −1.00 M; E and F ) in small pulmonary arteries from control and CH ASIC1 +/+ and ASIC1 −/− mice. Values are means ± SE; n = 5–7 animals/group. * P ≤ 0.05 vs. the control group; # P

Techniques Used: Mouse Assay

4) Product Images from "PICK1/calcineurin suppress ASIC1-mediated Ca2+ entry in rat pulmonary arterial smooth muscle cells"

Article Title: PICK1/calcineurin suppress ASIC1-mediated Ca2+ entry in rat pulmonary arterial smooth muscle cells

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00091.2015

PICK1 inhibits ASIC1-dependent SOCE. A : SOCE responses as determined by area under the curve (AUC) for PASMC pretreated with increasing concentrations of the PICK1 inhibitor, FSC231 (10–100 μM) for 24 h. Values are means ± SE;
Figure Legend Snippet: PICK1 inhibits ASIC1-dependent SOCE. A : SOCE responses as determined by area under the curve (AUC) for PASMC pretreated with increasing concentrations of the PICK1 inhibitor, FSC231 (10–100 μM) for 24 h. Values are means ± SE;

Techniques Used:

Store depletion increases the interaction and clustering between PICK1 and ASIC1. Representative confocal images ( A ) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 ( A ; red puncta) upon stimulation of store depletion [SD; Ca 2+
Figure Legend Snippet: Store depletion increases the interaction and clustering between PICK1 and ASIC1. Representative confocal images ( A ) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 ( A ; red puncta) upon stimulation of store depletion [SD; Ca 2+

Techniques Used:

Colocalization of PICK1 and acid-sensing ion channel 1 (ASIC1) in PASMC. A : representative confocal images of the Duolink PLA interaction between goat anti-ASIC1 and rabbit anti-PICK1 (red puncta). For negative controls, PASMC were incubated with each
Figure Legend Snippet: Colocalization of PICK1 and acid-sensing ion channel 1 (ASIC1) in PASMC. A : representative confocal images of the Duolink PLA interaction between goat anti-ASIC1 and rabbit anti-PICK1 (red puncta). For negative controls, PASMC were incubated with each

Techniques Used: Proximity Ligation Assay, Incubation

PICK1/Calcineurin inhibit PKA-stimulated SOCE. SOCE responses were determined by AUC in PASMC pretreated with the PICK1 inhibitor FSC231 (50 μM), calcineurin inhibitor cyclosporin A (CsA; 1 μM), ASIC1 inhibitor psalmotoxin 1 (PcTX1; 20
Figure Legend Snippet: PICK1/Calcineurin inhibit PKA-stimulated SOCE. SOCE responses were determined by AUC in PASMC pretreated with the PICK1 inhibitor FSC231 (50 μM), calcineurin inhibitor cyclosporin A (CsA; 1 μM), ASIC1 inhibitor psalmotoxin 1 (PcTX1; 20

Techniques Used:

PICK1 and calcineurin inhibit ASIC1 phosphorylation. PASMC were pretreated with vehicle; PKA activator forskolin (10 μM), PKA inhibitor KT 5720 (300 nM), PICK1 inhibitor FSC231 (50 μM), or calcineurin inhibitor CsA (1 μM). Endogenous
Figure Legend Snippet: PICK1 and calcineurin inhibit ASIC1 phosphorylation. PASMC were pretreated with vehicle; PKA activator forskolin (10 μM), PKA inhibitor KT 5720 (300 nM), PICK1 inhibitor FSC231 (50 μM), or calcineurin inhibitor CsA (1 μM). Endogenous

Techniques Used:

Summary of major findings. Ca 2+ depletion of the sarcoplasmic reticulum (SR) stimulates ASIC1-dependent SOCE (solid line) in PASMC. ASIC1-mediated SOCE is augmented by PKA-mediated phosphorylation and inhibited by PKC-mediated phosphorylation. Store depletion
Figure Legend Snippet: Summary of major findings. Ca 2+ depletion of the sarcoplasmic reticulum (SR) stimulates ASIC1-dependent SOCE (solid line) in PASMC. ASIC1-mediated SOCE is augmented by PKA-mediated phosphorylation and inhibited by PKC-mediated phosphorylation. Store depletion

Techniques Used:

Inhibition of PICK1 diminishes colocalization and clustering with ASIC1. Representative confocal images ( A ) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 ( A ; red puncta) in the absence or presence of the PICK1 inhibitor FSC231
Figure Legend Snippet: Inhibition of PICK1 diminishes colocalization and clustering with ASIC1. Representative confocal images ( A ) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 ( A ; red puncta) in the absence or presence of the PICK1 inhibitor FSC231

Techniques Used: Inhibition

PICK1 does not alter ASIC1 plasma membrane (PM) localization in PASMC. Representative Western blots ( A and B ) and summary data ( D ) showing the ratio of cell surface biotinylated ( A ) to total ( B ) ASIC1 expression following 24-h treatment with the PICK1
Figure Legend Snippet: PICK1 does not alter ASIC1 plasma membrane (PM) localization in PASMC. Representative Western blots ( A and B ) and summary data ( D ) showing the ratio of cell surface biotinylated ( A ) to total ( B ) ASIC1 expression following 24-h treatment with the PICK1

Techniques Used: Western Blot, Expressing

5) Product Images from "Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension"

Article Title: Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00269.2013

CH does not alter ASIC1 protein levels in small pulmonary arteries. A : representative Western blots of ASIC1 and GAPDH (20 μg protein/lane). B : summary data for Western blot analysis of ASIC1/GAPDH protein expression. Values are means ± SE; n values are indicated in bars.
Figure Legend Snippet: CH does not alter ASIC1 protein levels in small pulmonary arteries. A : representative Western blots of ASIC1 and GAPDH (20 μg protein/lane). B : summary data for Western blot analysis of ASIC1/GAPDH protein expression. Values are means ± SE; n values are indicated in bars.

Techniques Used: Western Blot, Expressing

ASIC1 protein expression in small pulmonary arteries. Smooth muscle α-actin (SM α-actin) and ASIC1 immunofluorescence in ASIC1 +/+ and ASIC1 −/− lung sections demonstrate the presence of ASIC1 in small pulmonary arteries (∼70–100 μm). Fluorescence images were digitally inverted using ImageJ to provide better contrast and visibility of immunofluorescence. The bottom images represent digitally zoomed fluorescence overlay images corresponding to the boxes in the images of smooth muscle α-actin (green; top ) and ASIC1 (red; middle ).
Figure Legend Snippet: ASIC1 protein expression in small pulmonary arteries. Smooth muscle α-actin (SM α-actin) and ASIC1 immunofluorescence in ASIC1 +/+ and ASIC1 −/− lung sections demonstrate the presence of ASIC1 in small pulmonary arteries (∼70–100 μm). Fluorescence images were digitally inverted using ImageJ to provide better contrast and visibility of immunofluorescence. The bottom images represent digitally zoomed fluorescence overlay images corresponding to the boxes in the images of smooth muscle α-actin (green; top ) and ASIC1 (red; middle ).

Techniques Used: Expressing, Immunofluorescence, Fluorescence

ASIC1 contributes to small pulmonary artery remodeling after CH. A : representative bright-field ( left ) or smooth muscle α-actin immunofluorescence ( right ) images of lung sections from control and CH ASIC1 +/+ and ASIC1 −/− mice. Bottom images show higher-magnification images of ∼10-μm arteries from CH ASIC1 +/+ and ASIC1 −/− mice. The arrow bottom middle image indicates an alveolar duct smooth muscle cell. Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. B –60 μm) from 20 random images/lung section. C : percent muscularization calculated as percent thresholded smooth muscle α-actin area divided by total arterial wall area. Values are means ± SE; n = 4 animals/group. * P ≤ 0.05 vs. the control group; # P
Figure Legend Snippet: ASIC1 contributes to small pulmonary artery remodeling after CH. A : representative bright-field ( left ) or smooth muscle α-actin immunofluorescence ( right ) images of lung sections from control and CH ASIC1 +/+ and ASIC1 −/− mice. Bottom images show higher-magnification images of ∼10-μm arteries from CH ASIC1 +/+ and ASIC1 −/− mice. The arrow bottom middle image indicates an alveolar duct smooth muscle cell. Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. B –60 μm) from 20 random images/lung section. C : percent muscularization calculated as percent thresholded smooth muscle α-actin area divided by total arterial wall area. Values are means ± SE; n = 4 animals/group. * P ≤ 0.05 vs. the control group; # P

Techniques Used: Immunofluorescence, Mouse Assay, Fluorescence

Restoration of acid-sensing ion channel 1 (ASIC1) in pulmonary arterial smooth muscle cells (PASMCs) from ASIC1 knockout (ASIC1 −/− ) mice rescues store-operated Ca 2+ entry (SOCE). A : RT-PCR for ASIC1 and β-actin in PASMCs from ASIC1 +/+ and ASIC1 −/− mice transfected with human (h)ASIC1. B : smooth muscle-22α (SM22α; green) and ASIC1 (red) immunofluorescence in PASMCs from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. C : summary data showing SOCE-induced changes (Δ) in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [expressed as changes in the 340-to-380-nm fluorescence ratio (ΔF 340 /F 380 )] in PASMCs isolated from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. All experiments were performed in the presence of cyclopiazonic acid (CPA; 10 μM) and diltiazem (50 μM). Values are means ± SE; numbers of animals ( n values) are indicated in bars. * P
Figure Legend Snippet: Restoration of acid-sensing ion channel 1 (ASIC1) in pulmonary arterial smooth muscle cells (PASMCs) from ASIC1 knockout (ASIC1 −/− ) mice rescues store-operated Ca 2+ entry (SOCE). A : RT-PCR for ASIC1 and β-actin in PASMCs from ASIC1 +/+ and ASIC1 −/− mice transfected with human (h)ASIC1. B : smooth muscle-22α (SM22α; green) and ASIC1 (red) immunofluorescence in PASMCs from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. C : summary data showing SOCE-induced changes (Δ) in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [expressed as changes in the 340-to-380-nm fluorescence ratio (ΔF 340 /F 380 )] in PASMCs isolated from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. All experiments were performed in the presence of cyclopiazonic acid (CPA; 10 μM) and diltiazem (50 μM). Values are means ± SE; numbers of animals ( n values) are indicated in bars. * P

Techniques Used: Knock-Out, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunofluorescence, Concentration Assay, Fluorescence, Isolation

ASIC1 contributes to enhanced receptor-mediated vasoconstriction after CH in small pulmonary arteries. A–F : vasoconstriction (percent baseline diameter; A , C , and E ) and changes in arterial wall [Ca 2+ ] i (ΔF 340 /F 380 ; B , D , and F ) to UTP (10 −7 to 3 × 10 −4 M; A and B ), endothelin-1 (ET-1; 10 −11 to 10 −7 M; C and D ), and depolarizing concentrations of KCl (10 −1.75 to 10 −1.00 M; E and F ) in small pulmonary arteries from control and CH ASIC1 +/+ and ASIC1 −/− mice. Values are means ± SE; n = 5–7 animals/group. * P ≤ 0.05 vs. the control group; # P
Figure Legend Snippet: ASIC1 contributes to enhanced receptor-mediated vasoconstriction after CH in small pulmonary arteries. A–F : vasoconstriction (percent baseline diameter; A , C , and E ) and changes in arterial wall [Ca 2+ ] i (ΔF 340 /F 380 ; B , D , and F ) to UTP (10 −7 to 3 × 10 −4 M; A and B ), endothelin-1 (ET-1; 10 −11 to 10 −7 M; C and D ), and depolarizing concentrations of KCl (10 −1.75 to 10 −1.00 M; E and F ) in small pulmonary arteries from control and CH ASIC1 +/+ and ASIC1 −/− mice. Values are means ± SE; n = 5–7 animals/group. * P ≤ 0.05 vs. the control group; # P

Techniques Used: Mouse Assay

6) Product Images from "Region specific contribution of ASIC2 to acidosis-and ischemia-induced neuronal injury"

Article Title: Region specific contribution of ASIC2 to acidosis-and ischemia-induced neuronal injury

Journal: Journal of Cerebral Blood Flow & Metabolism

doi: 10.1177/0271678X16630558

Differential surface trafficking of ASIC subunits in the brain. (a) Specificity of two ASIC1 antibodies. Brains from WT or ASIC1a −/− mice were blotted with a rabbit “DKG” IgG (left image) or a rabbit “MTY” antibody (right image). In these images, ASIC was detected in the green channel while tubulin was in red. (b–f) Representative Western blots (b) and quantification (c–f) showing surface expression of ASIC1a in WT and ASIC2 null (2KO) mice. Cerebellum (Cb), striatum (Str), hippocampus (Hp), and cortex (Cx) from 3- to 9-week-old mice were isolated. Biotinylation of acute brain tissue was performed as described in “Methods.” Surface and total proteins were blotted for ASIC1a (green) and tubulin (red). Note that ASIC2 null mice had reduced ASIC1a protein levels in all four brain regions, and that the surface ASIC1a level was not changed in cerebellum. In each experiment, the WT and knockout tissues were processed in parallel. ASIC: acid-sensing ion channel; WT: wild-type.
Figure Legend Snippet: Differential surface trafficking of ASIC subunits in the brain. (a) Specificity of two ASIC1 antibodies. Brains from WT or ASIC1a −/− mice were blotted with a rabbit “DKG” IgG (left image) or a rabbit “MTY” antibody (right image). In these images, ASIC was detected in the green channel while tubulin was in red. (b–f) Representative Western blots (b) and quantification (c–f) showing surface expression of ASIC1a in WT and ASIC2 null (2KO) mice. Cerebellum (Cb), striatum (Str), hippocampus (Hp), and cortex (Cx) from 3- to 9-week-old mice were isolated. Biotinylation of acute brain tissue was performed as described in “Methods.” Surface and total proteins were blotted for ASIC1a (green) and tubulin (red). Note that ASIC2 null mice had reduced ASIC1a protein levels in all four brain regions, and that the surface ASIC1a level was not changed in cerebellum. In each experiment, the WT and knockout tissues were processed in parallel. ASIC: acid-sensing ion channel; WT: wild-type.

Techniques Used: Mouse Assay, Western Blot, Expressing, Isolation, Knock-Out

7) Product Images from "miR-149 reduces while let-7 elevates ASIC1a expression in vitro"

Article Title: miR-149 reduces while let-7 elevates ASIC1a expression in vitro

Journal: International Journal of Physiology, Pathophysiology and Pharmacology

doi:

Effect of 30c and Let-7 on ASIC1a expression. CHO cells were transfected with ASIC1a-UTR together with the vector control, or expression constructs for miR-30c or let-7. Two days after transfection, cells were lysed and analyzed by Western blot using an anti-ASIC1 and an anti-b-tubulin antibodies. Representative blots and quantification of ASIC1a: tubulin ratio was shown. Asterisk indicates significant (p
Figure Legend Snippet: Effect of 30c and Let-7 on ASIC1a expression. CHO cells were transfected with ASIC1a-UTR together with the vector control, or expression constructs for miR-30c or let-7. Two days after transfection, cells were lysed and analyzed by Western blot using an anti-ASIC1 and an anti-b-tubulin antibodies. Representative blots and quantification of ASIC1a: tubulin ratio was shown. Asterisk indicates significant (p

Techniques Used: Expressing, Transfection, Plasmid Preparation, Construct, Western Blot

8) Product Images from "Region specific contribution of ASIC2 to acidosis-and ischemia-induced neuronal injury"

Article Title: Region specific contribution of ASIC2 to acidosis-and ischemia-induced neuronal injury

Journal: Journal of Cerebral Blood Flow & Metabolism

doi: 10.1177/0271678X16630558

Differential surface trafficking of ASIC subunits in the brain. (a) Specificity of two ASIC1 antibodies. Brains from WT or ASIC1a −/− mice were blotted with a rabbit “DKG” IgG (left image) or a rabbit “MTY” antibody (right image). In these images, ASIC was detected in the green channel while tubulin was in red. (b–f) Representative Western blots (b) and quantification (c–f) showing surface expression of ASIC1a in WT and ASIC2 null (2KO) mice. Cerebellum (Cb), striatum (Str), hippocampus (Hp), and cortex (Cx) from 3- to 9-week-old mice were isolated. Biotinylation of acute brain tissue was performed as described in “Methods.” Surface and total proteins were blotted for ASIC1a (green) and tubulin (red). Note that ASIC2 null mice had reduced ASIC1a protein levels in all four brain regions, and that the surface ASIC1a level was not changed in cerebellum. In each experiment, the WT and knockout tissues were processed in parallel. ASIC: acid-sensing ion channel; WT: wild-type.
Figure Legend Snippet: Differential surface trafficking of ASIC subunits in the brain. (a) Specificity of two ASIC1 antibodies. Brains from WT or ASIC1a −/− mice were blotted with a rabbit “DKG” IgG (left image) or a rabbit “MTY” antibody (right image). In these images, ASIC was detected in the green channel while tubulin was in red. (b–f) Representative Western blots (b) and quantification (c–f) showing surface expression of ASIC1a in WT and ASIC2 null (2KO) mice. Cerebellum (Cb), striatum (Str), hippocampus (Hp), and cortex (Cx) from 3- to 9-week-old mice were isolated. Biotinylation of acute brain tissue was performed as described in “Methods.” Surface and total proteins were blotted for ASIC1a (green) and tubulin (red). Note that ASIC2 null mice had reduced ASIC1a protein levels in all four brain regions, and that the surface ASIC1a level was not changed in cerebellum. In each experiment, the WT and knockout tissues were processed in parallel. ASIC: acid-sensing ion channel; WT: wild-type.

Techniques Used: Mouse Assay, Western Blot, Expressing, Isolation, Knock-Out

9) Product Images from "PICK1/calcineurin suppress ASIC1-mediated Ca2+ entry in rat pulmonary arterial smooth muscle cells"

Article Title: PICK1/calcineurin suppress ASIC1-mediated Ca2+ entry in rat pulmonary arterial smooth muscle cells

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00091.2015

PICK1 inhibits ASIC1-dependent SOCE. A : SOCE responses as determined by area under the curve (AUC) for PASMC pretreated with increasing concentrations of the PICK1 inhibitor, FSC231 (10–100 μM) for 24 h. Values are means ± SE;
Figure Legend Snippet: PICK1 inhibits ASIC1-dependent SOCE. A : SOCE responses as determined by area under the curve (AUC) for PASMC pretreated with increasing concentrations of the PICK1 inhibitor, FSC231 (10–100 μM) for 24 h. Values are means ± SE;

Techniques Used:

Store depletion increases the interaction and clustering between PICK1 and ASIC1. Representative confocal images ( A ) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 ( A ; red puncta) upon stimulation of store depletion [SD; Ca 2+
Figure Legend Snippet: Store depletion increases the interaction and clustering between PICK1 and ASIC1. Representative confocal images ( A ) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 ( A ; red puncta) upon stimulation of store depletion [SD; Ca 2+

Techniques Used:

Colocalization of PICK1 and acid-sensing ion channel 1 (ASIC1) in PASMC. A : representative confocal images of the Duolink PLA interaction between goat anti-ASIC1 and rabbit anti-PICK1 (red puncta). For negative controls, PASMC were incubated with each
Figure Legend Snippet: Colocalization of PICK1 and acid-sensing ion channel 1 (ASIC1) in PASMC. A : representative confocal images of the Duolink PLA interaction between goat anti-ASIC1 and rabbit anti-PICK1 (red puncta). For negative controls, PASMC were incubated with each

Techniques Used: Proximity Ligation Assay, Incubation

PICK1/Calcineurin inhibit PKA-stimulated SOCE. SOCE responses were determined by AUC in PASMC pretreated with the PICK1 inhibitor FSC231 (50 μM), calcineurin inhibitor cyclosporin A (CsA; 1 μM), ASIC1 inhibitor psalmotoxin 1 (PcTX1; 20
Figure Legend Snippet: PICK1/Calcineurin inhibit PKA-stimulated SOCE. SOCE responses were determined by AUC in PASMC pretreated with the PICK1 inhibitor FSC231 (50 μM), calcineurin inhibitor cyclosporin A (CsA; 1 μM), ASIC1 inhibitor psalmotoxin 1 (PcTX1; 20

Techniques Used:

PICK1 and calcineurin inhibit ASIC1 phosphorylation. PASMC were pretreated with vehicle; PKA activator forskolin (10 μM), PKA inhibitor KT 5720 (300 nM), PICK1 inhibitor FSC231 (50 μM), or calcineurin inhibitor CsA (1 μM). Endogenous
Figure Legend Snippet: PICK1 and calcineurin inhibit ASIC1 phosphorylation. PASMC were pretreated with vehicle; PKA activator forskolin (10 μM), PKA inhibitor KT 5720 (300 nM), PICK1 inhibitor FSC231 (50 μM), or calcineurin inhibitor CsA (1 μM). Endogenous

Techniques Used:

Summary of major findings. Ca 2+ depletion of the sarcoplasmic reticulum (SR) stimulates ASIC1-dependent SOCE (solid line) in PASMC. ASIC1-mediated SOCE is augmented by PKA-mediated phosphorylation and inhibited by PKC-mediated phosphorylation. Store depletion
Figure Legend Snippet: Summary of major findings. Ca 2+ depletion of the sarcoplasmic reticulum (SR) stimulates ASIC1-dependent SOCE (solid line) in PASMC. ASIC1-mediated SOCE is augmented by PKA-mediated phosphorylation and inhibited by PKC-mediated phosphorylation. Store depletion

Techniques Used:

Inhibition of PICK1 diminishes colocalization and clustering with ASIC1. Representative confocal images ( A ) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 ( A ; red puncta) in the absence or presence of the PICK1 inhibitor FSC231
Figure Legend Snippet: Inhibition of PICK1 diminishes colocalization and clustering with ASIC1. Representative confocal images ( A ) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 ( A ; red puncta) in the absence or presence of the PICK1 inhibitor FSC231

Techniques Used: Inhibition

PICK1 does not alter ASIC1 plasma membrane (PM) localization in PASMC. Representative Western blots ( A and B ) and summary data ( D ) showing the ratio of cell surface biotinylated ( A ) to total ( B ) ASIC1 expression following 24-h treatment with the PICK1
Figure Legend Snippet: PICK1 does not alter ASIC1 plasma membrane (PM) localization in PASMC. Representative Western blots ( A and B ) and summary data ( D ) showing the ratio of cell surface biotinylated ( A ) to total ( B ) ASIC1 expression following 24-h treatment with the PICK1

Techniques Used: Western Blot, Expressing

10) Product Images from "Human ASIC1a mediates stronger acid-induced responses as compared with mouse ASIC1a"

Article Title: Human ASIC1a mediates stronger acid-induced responses as compared with mouse ASIC1a

Journal: The FASEB Journal

doi: 10.1096/fj.201701367R

hASIC1a and mASIC1a S285P elicit a larger acid-activated calcium increase. CHO cells were transfected with various ASIC1 constructs, as indicated together with cameleon YC3.60, a ratiometric calcium reporter. FRET imaging and spectral unmixing were performed, as described in Materials and Methods. A ) Representative images showing the fluorescent images of transfected cells (leftmost) and ratiometric images (right 2 sets) before, at peak, and after stimulation with a pH 6.5 solution. For ratio images, red and blue indicate high and low FRET, respectively. B ) Average time-dependent changes in FRET (YFP/CFP ratio) of cells expressing mASIC1a, hASIC1a, and mASIC1a S285P. C ) Quantification of peak change in FRET (ΔFRET) of cells expressing the 3 different constructs. Bar numbers indicate total number of cells analyzed ( B, C ). Asterisk indicates differences from mASIC1a (ANOVA). Numbers in bars indicate number of repeats.
Figure Legend Snippet: hASIC1a and mASIC1a S285P elicit a larger acid-activated calcium increase. CHO cells were transfected with various ASIC1 constructs, as indicated together with cameleon YC3.60, a ratiometric calcium reporter. FRET imaging and spectral unmixing were performed, as described in Materials and Methods. A ) Representative images showing the fluorescent images of transfected cells (leftmost) and ratiometric images (right 2 sets) before, at peak, and after stimulation with a pH 6.5 solution. For ratio images, red and blue indicate high and low FRET, respectively. B ) Average time-dependent changes in FRET (YFP/CFP ratio) of cells expressing mASIC1a, hASIC1a, and mASIC1a S285P. C ) Quantification of peak change in FRET (ΔFRET) of cells expressing the 3 different constructs. Bar numbers indicate total number of cells analyzed ( B, C ). Asterisk indicates differences from mASIC1a (ANOVA). Numbers in bars indicate number of repeats.

Techniques Used: Transfection, Construct, Imaging, Expressing

Expression of ASIC1 and -2 in human cortical tissue. A ) A typical set of images and traces showing the identification of epileptic foci in 1 patient. Diagram illustrates the layout of the recording electrodes (top left), whereas the MRIs on the right illustrate the location of the epileptic foci (indicated by a red dot) in hippocampus. EEG traces from neocortex and hippocampus: hippocampus exhibited epileptic patterns of activities, which were absent in cortical traces (bottom). Note that the cortical samples were used in this study for comparing ASIC expression between human and mouse brain. B ). Human and mouse tissues were not statistically different in the relative ratio of ASIC1a:ASIC2a ( P > 0.05, either by Student’s t test or Wilcoxon signed rank test) (middle). The relative ASIC2b:ASIC2a ratio was significantly lower in human tissue. P = 0.006 (Student’s t test) (right). C ) Representative Western blot images and quantification showing ASIC1a levels in membrane (left). Membrane fractions from human and mouse cortical tissues were prepared, as described in Materials and Methods. Total lysate, membrane fraction, and cytosol fraction were blotted for ASIC1a, N-cadherin, and β-tubulin III. The latter 2 proteins served as controls for the quality of the preparation. Right: quantification of relative membrane:total ASIC1a ratios. Asterisks ( B , C ) indicate significant differences between mouse and human. Numbers in bars indicate number of repeats.
Figure Legend Snippet: Expression of ASIC1 and -2 in human cortical tissue. A ) A typical set of images and traces showing the identification of epileptic foci in 1 patient. Diagram illustrates the layout of the recording electrodes (top left), whereas the MRIs on the right illustrate the location of the epileptic foci (indicated by a red dot) in hippocampus. EEG traces from neocortex and hippocampus: hippocampus exhibited epileptic patterns of activities, which were absent in cortical traces (bottom). Note that the cortical samples were used in this study for comparing ASIC expression between human and mouse brain. B ). Human and mouse tissues were not statistically different in the relative ratio of ASIC1a:ASIC2a ( P > 0.05, either by Student’s t test or Wilcoxon signed rank test) (middle). The relative ASIC2b:ASIC2a ratio was significantly lower in human tissue. P = 0.006 (Student’s t test) (right). C ) Representative Western blot images and quantification showing ASIC1a levels in membrane (left). Membrane fractions from human and mouse cortical tissues were prepared, as described in Materials and Methods. Total lysate, membrane fraction, and cytosol fraction were blotted for ASIC1a, N-cadherin, and β-tubulin III. The latter 2 proteins served as controls for the quality of the preparation. Right: quantification of relative membrane:total ASIC1a ratios. Asterisks ( B , C ) indicate significant differences between mouse and human. Numbers in bars indicate number of repeats.

Techniques Used: Expressing, Western Blot

11) Product Images from "ASIC1-mediated calcium entry stimulates NFATc3 nuclear translocation via PICK1 coupling in pulmonary arterial smooth muscle cells"

Article Title: ASIC1-mediated calcium entry stimulates NFATc3 nuclear translocation via PICK1 coupling in pulmonary arterial smooth muscle cells

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

doi: 10.1152/ajplung.00040.2016

Knockout of ASIC1 does not alter NFAT, Orai1, or TRPC1 mRNA and protein levels in PASMC. Fold change (from calibrator) in NFATc2 ( A ), NFATc3 ( B ), Orai1 ( C ), and TRPC1 ( D ) mRNA expression in PASMC from ASIC1 +/+ and ASIC1 −/− mice. β-Actin
Figure Legend Snippet: Knockout of ASIC1 does not alter NFAT, Orai1, or TRPC1 mRNA and protein levels in PASMC. Fold change (from calibrator) in NFATc2 ( A ), NFATc3 ( B ), Orai1 ( C ), and TRPC1 ( D ) mRNA expression in PASMC from ASIC1 +/+ and ASIC1 −/− mice. β-Actin

Techniques Used: Knock-Out, Expressing, Mouse Assay

ASIC1 Ca 2+ influx contributes to ET-1-induced NFATc3 nuclear import. A : mouse PASMC from ASIC1 +/+ or ASIC1 −/− mice were transfected with NFATc3-enhanced green fluorescent protein (EGFP). EGFP nuclear (Fn)/cytosolic fluorescence (Fc) was
Figure Legend Snippet: ASIC1 Ca 2+ influx contributes to ET-1-induced NFATc3 nuclear import. A : mouse PASMC from ASIC1 +/+ or ASIC1 −/− mice were transfected with NFATc3-enhanced green fluorescent protein (EGFP). EGFP nuclear (Fn)/cytosolic fluorescence (Fc) was

Techniques Used: Mouse Assay, Transfection, Fluorescence

ASIC1 contributes to CH-induced NFATc3 nuclear accumulation. Representative images ( A ) and summary data ( B ) showing NFATc3 nuclear translocation (white) was assessed by % NFATc3 colocalization (red) with SYTOX nuclear stain (green) in smooth muscle α-actin-positive
Figure Legend Snippet: ASIC1 contributes to CH-induced NFATc3 nuclear accumulation. Representative images ( A ) and summary data ( B ) showing NFATc3 nuclear translocation (white) was assessed by % NFATc3 colocalization (red) with SYTOX nuclear stain (green) in smooth muscle α-actin-positive

Techniques Used: Translocation Assay, Staining

Summary of major findings. CH, presumably through ET-1, increases Ca 2+ influx through ASIC1 in PASMC. Elevated intracellular Ca 2+ levels leads to calcineurin-mediated dephosphorylation of NFATc3, allowing its nuclear import. PICK1 provides the scaffold
Figure Legend Snippet: Summary of major findings. CH, presumably through ET-1, increases Ca 2+ influx through ASIC1 in PASMC. Elevated intracellular Ca 2+ levels leads to calcineurin-mediated dephosphorylation of NFATc3, allowing its nuclear import. PICK1 provides the scaffold

Techniques Used: De-Phosphorylation Assay

Chronic hypoxia (CH)-induced increases in basal pulmonary arterial intracellular Ca 2+ concentration ([Ca 2+ ] i ) and store-operated calcium entry (SOCE) are ASIC1 dependent. A : basal arterial wall [Ca 2+ ] i ratios (F 340 /F 380 ) in small pulmonary arteries from
Figure Legend Snippet: Chronic hypoxia (CH)-induced increases in basal pulmonary arterial intracellular Ca 2+ concentration ([Ca 2+ ] i ) and store-operated calcium entry (SOCE) are ASIC1 dependent. A : basal arterial wall [Ca 2+ ] i ratios (F 340 /F 380 ) in small pulmonary arteries from

Techniques Used: Concentration Assay

ASIC1 contributes to ET-1-mediated vasoconstriction in isolated, pressurized small pulmonary arteries. Vasoconstriction (percent baseline diameter, A and B ) and changes in arterial wall [Ca 2+ ] i (ΔF 340 /F 380 , C and D ) in response to endothelin-1
Figure Legend Snippet: ASIC1 contributes to ET-1-mediated vasoconstriction in isolated, pressurized small pulmonary arteries. Vasoconstriction (percent baseline diameter, A and B ) and changes in arterial wall [Ca 2+ ] i (ΔF 340 /F 380 , C and D ) in response to endothelin-1

Techniques Used: Isolation

ASIC1 contributes to ET-1-induced Ca 2+ influx in mouse PASMC. A : representative traces showing the change in fura-2 ratio. B : summary data of area under the curve (AUC) in response to ET-1 in mouse pulmonary arterial smooth muscle cell (PASMC) from ASIC1
Figure Legend Snippet: ASIC1 contributes to ET-1-induced Ca 2+ influx in mouse PASMC. A : representative traces showing the change in fura-2 ratio. B : summary data of area under the curve (AUC) in response to ET-1 in mouse pulmonary arterial smooth muscle cell (PASMC) from ASIC1

Techniques Used:

Colocalization of ASIC1, PICK1, and calcineurin B in mouse PASMC. Representative confocal images of the Duolink PLA interaction (red puncta) between ASIC1-PICK1 ( left ), ASIC1-calcineurin B ( middle ), and PICK1-calcineurin B ( right ) in PASMC from ASIC1
Figure Legend Snippet: Colocalization of ASIC1, PICK1, and calcineurin B in mouse PASMC. Representative confocal images of the Duolink PLA interaction (red puncta) between ASIC1-PICK1 ( left ), ASIC1-calcineurin B ( middle ), and PICK1-calcineurin B ( right ) in PASMC from ASIC1

Techniques Used: Proximity Ligation Assay

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    Santa Cruz Biotechnology goat anti asic1
    Effect of 30c and Let-7 on ASIC1a expression. CHO cells were transfected with ASIC1a-UTR together with the vector control, or expression constructs for miR-30c or let-7. Two days after transfection, cells were lysed and analyzed by Western blot using an <t>anti-ASIC1</t> and an anti-b-tubulin antibodies. Representative blots and quantification of ASIC1a: tubulin ratio was shown. Asterisk indicates significant (p
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    Effect of 30c and Let-7 on ASIC1a expression. CHO cells were transfected with ASIC1a-UTR together with the vector control, or expression constructs for miR-30c or let-7. Two days after transfection, cells were lysed and analyzed by Western blot using an anti-ASIC1 and an anti-b-tubulin antibodies. Representative blots and quantification of ASIC1a: tubulin ratio was shown. Asterisk indicates significant (p

    Journal: International Journal of Physiology, Pathophysiology and Pharmacology

    Article Title: miR-149 reduces while let-7 elevates ASIC1a expression in vitro

    doi:

    Figure Lengend Snippet: Effect of 30c and Let-7 on ASIC1a expression. CHO cells were transfected with ASIC1a-UTR together with the vector control, or expression constructs for miR-30c or let-7. Two days after transfection, cells were lysed and analyzed by Western blot using an anti-ASIC1 and an anti-b-tubulin antibodies. Representative blots and quantification of ASIC1a: tubulin ratio was shown. Asterisk indicates significant (p

    Article Snippet: ASIC antibodies used: a rabbit anti-ASIC1 antibody [ ] and a goat anti-ASIC1 (Santa Cruz SC-13905).

    Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Western Blot

    hASIC1a and mASIC1a S285P elicit a larger acid-activated calcium increase. CHO cells were transfected with various ASIC1 constructs, as indicated together with cameleon YC3.60, a ratiometric calcium reporter. FRET imaging and spectral unmixing were performed, as described in Materials and Methods. A ) Representative images showing the fluorescent images of transfected cells (leftmost) and ratiometric images (right 2 sets) before, at peak, and after stimulation with a pH 6.5 solution. For ratio images, red and blue indicate high and low FRET, respectively. B ) Average time-dependent changes in FRET (YFP/CFP ratio) of cells expressing mASIC1a, hASIC1a, and mASIC1a S285P. C ) Quantification of peak change in FRET (ΔFRET) of cells expressing the 3 different constructs. Bar numbers indicate total number of cells analyzed ( B, C ). Asterisk indicates differences from mASIC1a (ANOVA). Numbers in bars indicate number of repeats.

    Journal: The FASEB Journal

    Article Title: Human ASIC1a mediates stronger acid-induced responses as compared with mouse ASIC1a

    doi: 10.1096/fj.201701367R

    Figure Lengend Snippet: hASIC1a and mASIC1a S285P elicit a larger acid-activated calcium increase. CHO cells were transfected with various ASIC1 constructs, as indicated together with cameleon YC3.60, a ratiometric calcium reporter. FRET imaging and spectral unmixing were performed, as described in Materials and Methods. A ) Representative images showing the fluorescent images of transfected cells (leftmost) and ratiometric images (right 2 sets) before, at peak, and after stimulation with a pH 6.5 solution. For ratio images, red and blue indicate high and low FRET, respectively. B ) Average time-dependent changes in FRET (YFP/CFP ratio) of cells expressing mASIC1a, hASIC1a, and mASIC1a S285P. C ) Quantification of peak change in FRET (ΔFRET) of cells expressing the 3 different constructs. Bar numbers indicate total number of cells analyzed ( B, C ). Asterisk indicates differences from mASIC1a (ANOVA). Numbers in bars indicate number of repeats.

    Article Snippet: ASIC antibodies used the following: a rabbit anti-ASIC1 antibody ( ) and a goat anti-ASIC1 (SC-13905; Santa Cruz, Biotechnology, Dallas, TX, USA) and rabbit anti-ASIC2 ( ).

    Techniques: Transfection, Construct, Imaging, Expressing

    Expression of ASIC1 and -2 in human cortical tissue. A ) A typical set of images and traces showing the identification of epileptic foci in 1 patient. Diagram illustrates the layout of the recording electrodes (top left), whereas the MRIs on the right illustrate the location of the epileptic foci (indicated by a red dot) in hippocampus. EEG traces from neocortex and hippocampus: hippocampus exhibited epileptic patterns of activities, which were absent in cortical traces (bottom). Note that the cortical samples were used in this study for comparing ASIC expression between human and mouse brain. B ). Human and mouse tissues were not statistically different in the relative ratio of ASIC1a:ASIC2a ( P > 0.05, either by Student’s t test or Wilcoxon signed rank test) (middle). The relative ASIC2b:ASIC2a ratio was significantly lower in human tissue. P = 0.006 (Student’s t test) (right). C ) Representative Western blot images and quantification showing ASIC1a levels in membrane (left). Membrane fractions from human and mouse cortical tissues were prepared, as described in Materials and Methods. Total lysate, membrane fraction, and cytosol fraction were blotted for ASIC1a, N-cadherin, and β-tubulin III. The latter 2 proteins served as controls for the quality of the preparation. Right: quantification of relative membrane:total ASIC1a ratios. Asterisks ( B , C ) indicate significant differences between mouse and human. Numbers in bars indicate number of repeats.

    Journal: The FASEB Journal

    Article Title: Human ASIC1a mediates stronger acid-induced responses as compared with mouse ASIC1a

    doi: 10.1096/fj.201701367R

    Figure Lengend Snippet: Expression of ASIC1 and -2 in human cortical tissue. A ) A typical set of images and traces showing the identification of epileptic foci in 1 patient. Diagram illustrates the layout of the recording electrodes (top left), whereas the MRIs on the right illustrate the location of the epileptic foci (indicated by a red dot) in hippocampus. EEG traces from neocortex and hippocampus: hippocampus exhibited epileptic patterns of activities, which were absent in cortical traces (bottom). Note that the cortical samples were used in this study for comparing ASIC expression between human and mouse brain. B ). Human and mouse tissues were not statistically different in the relative ratio of ASIC1a:ASIC2a ( P > 0.05, either by Student’s t test or Wilcoxon signed rank test) (middle). The relative ASIC2b:ASIC2a ratio was significantly lower in human tissue. P = 0.006 (Student’s t test) (right). C ) Representative Western blot images and quantification showing ASIC1a levels in membrane (left). Membrane fractions from human and mouse cortical tissues were prepared, as described in Materials and Methods. Total lysate, membrane fraction, and cytosol fraction were blotted for ASIC1a, N-cadherin, and β-tubulin III. The latter 2 proteins served as controls for the quality of the preparation. Right: quantification of relative membrane:total ASIC1a ratios. Asterisks ( B , C ) indicate significant differences between mouse and human. Numbers in bars indicate number of repeats.

    Article Snippet: ASIC antibodies used the following: a rabbit anti-ASIC1 antibody ( ) and a goat anti-ASIC1 (SC-13905; Santa Cruz, Biotechnology, Dallas, TX, USA) and rabbit anti-ASIC2 ( ).

    Techniques: Expressing, Western Blot

    CH does not alter ASIC1 protein levels in small pulmonary arteries. A : representative Western blots of ASIC1 and GAPDH (20 μg protein/lane). B : summary data for Western blot analysis of ASIC1/GAPDH protein expression. Values are means ± SE; n values are indicated in bars.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension

    doi: 10.1152/ajpheart.00269.2013

    Figure Lengend Snippet: CH does not alter ASIC1 protein levels in small pulmonary arteries. A : representative Western blots of ASIC1 and GAPDH (20 μg protein/lane). B : summary data for Western blot analysis of ASIC1/GAPDH protein expression. Values are means ± SE; n values are indicated in bars.

    Article Snippet: Transfected and nontransfected PASMCs from ASIC1+/+ and ASIC1−/− mice were fixed with 2% paraformaldehyde and incubated overnight at 4°C with rabbit anti-smooth muscle-22α (1:200, Abcam) and goat anti-ASIC1 (1:50, Santa Cruz Biotechnology).

    Techniques: Western Blot, Expressing

    ASIC1 protein expression in small pulmonary arteries. Smooth muscle α-actin (SM α-actin) and ASIC1 immunofluorescence in ASIC1 +/+ and ASIC1 −/− lung sections demonstrate the presence of ASIC1 in small pulmonary arteries (∼70–100 μm). Fluorescence images were digitally inverted using ImageJ to provide better contrast and visibility of immunofluorescence. The bottom images represent digitally zoomed fluorescence overlay images corresponding to the boxes in the images of smooth muscle α-actin (green; top ) and ASIC1 (red; middle ).

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension

    doi: 10.1152/ajpheart.00269.2013

    Figure Lengend Snippet: ASIC1 protein expression in small pulmonary arteries. Smooth muscle α-actin (SM α-actin) and ASIC1 immunofluorescence in ASIC1 +/+ and ASIC1 −/− lung sections demonstrate the presence of ASIC1 in small pulmonary arteries (∼70–100 μm). Fluorescence images were digitally inverted using ImageJ to provide better contrast and visibility of immunofluorescence. The bottom images represent digitally zoomed fluorescence overlay images corresponding to the boxes in the images of smooth muscle α-actin (green; top ) and ASIC1 (red; middle ).

    Article Snippet: Transfected and nontransfected PASMCs from ASIC1+/+ and ASIC1−/− mice were fixed with 2% paraformaldehyde and incubated overnight at 4°C with rabbit anti-smooth muscle-22α (1:200, Abcam) and goat anti-ASIC1 (1:50, Santa Cruz Biotechnology).

    Techniques: Expressing, Immunofluorescence, Fluorescence

    ASIC1 contributes to small pulmonary artery remodeling after CH. A : representative bright-field ( left ) or smooth muscle α-actin immunofluorescence ( right ) images of lung sections from control and CH ASIC1 +/+ and ASIC1 −/− mice. Bottom images show higher-magnification images of ∼10-μm arteries from CH ASIC1 +/+ and ASIC1 −/− mice. The arrow bottom middle image indicates an alveolar duct smooth muscle cell. Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. B –60 μm) from 20 random images/lung section. C : percent muscularization calculated as percent thresholded smooth muscle α-actin area divided by total arterial wall area. Values are means ± SE; n = 4 animals/group. * P ≤ 0.05 vs. the control group; # P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension

    doi: 10.1152/ajpheart.00269.2013

    Figure Lengend Snippet: ASIC1 contributes to small pulmonary artery remodeling after CH. A : representative bright-field ( left ) or smooth muscle α-actin immunofluorescence ( right ) images of lung sections from control and CH ASIC1 +/+ and ASIC1 −/− mice. Bottom images show higher-magnification images of ∼10-μm arteries from CH ASIC1 +/+ and ASIC1 −/− mice. The arrow bottom middle image indicates an alveolar duct smooth muscle cell. Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. B –60 μm) from 20 random images/lung section. C : percent muscularization calculated as percent thresholded smooth muscle α-actin area divided by total arterial wall area. Values are means ± SE; n = 4 animals/group. * P ≤ 0.05 vs. the control group; # P

    Article Snippet: Transfected and nontransfected PASMCs from ASIC1+/+ and ASIC1−/− mice were fixed with 2% paraformaldehyde and incubated overnight at 4°C with rabbit anti-smooth muscle-22α (1:200, Abcam) and goat anti-ASIC1 (1:50, Santa Cruz Biotechnology).

    Techniques: Immunofluorescence, Mouse Assay, Fluorescence

    Restoration of acid-sensing ion channel 1 (ASIC1) in pulmonary arterial smooth muscle cells (PASMCs) from ASIC1 knockout (ASIC1 −/− ) mice rescues store-operated Ca 2+ entry (SOCE). A : RT-PCR for ASIC1 and β-actin in PASMCs from ASIC1 +/+ and ASIC1 −/− mice transfected with human (h)ASIC1. B : smooth muscle-22α (SM22α; green) and ASIC1 (red) immunofluorescence in PASMCs from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. C : summary data showing SOCE-induced changes (Δ) in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [expressed as changes in the 340-to-380-nm fluorescence ratio (ΔF 340 /F 380 )] in PASMCs isolated from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. All experiments were performed in the presence of cyclopiazonic acid (CPA; 10 μM) and diltiazem (50 μM). Values are means ± SE; numbers of animals ( n values) are indicated in bars. * P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension

    doi: 10.1152/ajpheart.00269.2013

    Figure Lengend Snippet: Restoration of acid-sensing ion channel 1 (ASIC1) in pulmonary arterial smooth muscle cells (PASMCs) from ASIC1 knockout (ASIC1 −/− ) mice rescues store-operated Ca 2+ entry (SOCE). A : RT-PCR for ASIC1 and β-actin in PASMCs from ASIC1 +/+ and ASIC1 −/− mice transfected with human (h)ASIC1. B : smooth muscle-22α (SM22α; green) and ASIC1 (red) immunofluorescence in PASMCs from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. C : summary data showing SOCE-induced changes (Δ) in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [expressed as changes in the 340-to-380-nm fluorescence ratio (ΔF 340 /F 380 )] in PASMCs isolated from ASIC1 +/+ and ASIC1 −/− mice. PASMCs from ASIC1 −/− mice were additionally transfected with hASIC1. All experiments were performed in the presence of cyclopiazonic acid (CPA; 10 μM) and diltiazem (50 μM). Values are means ± SE; numbers of animals ( n values) are indicated in bars. * P

    Article Snippet: Transfected and nontransfected PASMCs from ASIC1+/+ and ASIC1−/− mice were fixed with 2% paraformaldehyde and incubated overnight at 4°C with rabbit anti-smooth muscle-22α (1:200, Abcam) and goat anti-ASIC1 (1:50, Santa Cruz Biotechnology).

    Techniques: Knock-Out, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunofluorescence, Concentration Assay, Fluorescence, Isolation

    ASIC1 contributes to enhanced receptor-mediated vasoconstriction after CH in small pulmonary arteries. A–F : vasoconstriction (percent baseline diameter; A , C , and E ) and changes in arterial wall [Ca 2+ ] i (ΔF 340 /F 380 ; B , D , and F ) to UTP (10 −7 to 3 × 10 −4 M; A and B ), endothelin-1 (ET-1; 10 −11 to 10 −7 M; C and D ), and depolarizing concentrations of KCl (10 −1.75 to 10 −1.00 M; E and F ) in small pulmonary arteries from control and CH ASIC1 +/+ and ASIC1 −/− mice. Values are means ± SE; n = 5–7 animals/group. * P ≤ 0.05 vs. the control group; # P

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension

    doi: 10.1152/ajpheart.00269.2013

    Figure Lengend Snippet: ASIC1 contributes to enhanced receptor-mediated vasoconstriction after CH in small pulmonary arteries. A–F : vasoconstriction (percent baseline diameter; A , C , and E ) and changes in arterial wall [Ca 2+ ] i (ΔF 340 /F 380 ; B , D , and F ) to UTP (10 −7 to 3 × 10 −4 M; A and B ), endothelin-1 (ET-1; 10 −11 to 10 −7 M; C and D ), and depolarizing concentrations of KCl (10 −1.75 to 10 −1.00 M; E and F ) in small pulmonary arteries from control and CH ASIC1 +/+ and ASIC1 −/− mice. Values are means ± SE; n = 5–7 animals/group. * P ≤ 0.05 vs. the control group; # P

    Article Snippet: Transfected and nontransfected PASMCs from ASIC1+/+ and ASIC1−/− mice were fixed with 2% paraformaldehyde and incubated overnight at 4°C with rabbit anti-smooth muscle-22α (1:200, Abcam) and goat anti-ASIC1 (1:50, Santa Cruz Biotechnology).

    Techniques: Mouse Assay