Journal: The Journal of Neuroscience

Article Title: Acid-Sensing Ion Channel 2 Is Important for Retinal Function and Protects against Light-Induced Retinal Degeneration

doi: 10.1523/JNEUROSCI.4698-03.2004

Figure Lengend Snippet: ASIC transcripts and protein in the mouse retina. A–D, In situ hybridization with retinas from 3-month-old ASIC2+/+ (A, C) and ASIC2–/– (B, D) mice with ASIC2a (A, B) or ASIC2b (C, D) probes. In wild-type mice, ASIC2a and ASIC2b mRNAs are expressed in the outer nuclear layer (ONL) and in some cells of the distal and proximal portion of the inner nuclear layer (INL; arrows). Expression of both transcripts was reduced in ASIC2 null mice. Similar results were obtained with three antisense oligonucleotides for each transcript. Both omission of the oligonucleotide and incubation with a DIG-labeled oligonucleotide unrelated to ASIC2 did not yield any labeling. OS, Outer segment; IS, inner segment; OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer. E, Western blot with an antibody directed against the ASIC2a NH2 terminus confirmed the presence of ASIC2a in the retina. COS, A total of 6 μg of homogenate from ASIC2a-transfected COS cells. +/+ and –/–, A total of 20 μg of retina homogenate from ASIC2+/+ or ASIC2–/– mice. Although a protein of ∼60 kDa is labeled in ASIC2+/+ mice, neither the 60 kDa protein nor the 7 kDa shorter protein for which the targeted ASIC2 transcript codes is detected in ASIC2–/– mice. The smaller apparent molecular weight of ASIC2 heterologously expressed in COS is probably attributable to a lower glycosylation level. The proteins detected on the blot are recognized by the anti-ASIC2 antibody, because omission of the primary antibody abolished their labeling (data not shown). The anti-ASIC2 antibodies that we prepared as well as the commercialized anti ASIC2a-antibodies obtained from Alomone Labs, Chemicon, and Alpha Diagnostics directed against NH2 terminal peptides all cross-react strongly with another protein slightly bigger than ASIC2a in ASIC2–/– mice.

Article Snippet: The anti-ASIC2 antibodies that we prepared as well as the commercialized anti ASIC2a-antibodies obtained from Alomone Labs, Chemicon, and Alpha Diagnostics directed against NH2 terminal peptides all cross-react strongly with another protein slightly bigger than ASIC2a in ASIC2 –/– mice.

Techniques: In Situ Hybridization, Expressing, Incubation, Labeling, Western Blot, Transfection, Molecular Weight

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Altered hippocampal TFCP2 and ASIC2a expression with glucose hypometabolism in patients with TLE. ( a ) Patient 4’s pre-surgical assessment results: magnetic resonance imaging (left) was negative, electroencephalography (middle) showed spike waves in the temporal lobe, and fluorodeoxyglucose positron emission tomography (right) revealed hypometabolic lesions in the right hippocampus. ( b ) Representative western blot assays of hippocampal TFCP2 and ASIC2a expression in patients with TLE (n = 13) and control patients (n = 10). β-actin was used as a loading control. ( c , d ) Normalised densitometry bar graphs of TFCP2 and ASIC2a for the control subjects and patients with TLE. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P < 0.05, **P < 0.01 compared to controls. Uncropped western blot images are shown in Supplementary Fig. . Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; TLE: temporal lobe epilepsy.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Expressing, Magnetic Resonance Imaging, Positron Emission Tomography, Western Blot, Control

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Altered hippocampal TFCP2 and ASIC2a expression with glucose hypometabolism in pilocarpine-treated rats. ( a ) Representative coronal view microscopic positron emission tomography images in the different phases of epileptogenesis. ( b ) Hippocampal glucose uptake in the different phases after pilocarpine injection. ( c ) Representative western blot assays of hippocampal TFCP2 and ASIC2a expression at different post-seizure time points. ( d ) Bar graph showing decreased hippocampal TFCP2 expression in pilocarpine-treated rats. ( e ) Bar graph showing increased hippocampal ASIC2a expression in pilocarpine-treated rats. The experiments were repeated at least 3 times with at least 3 rats in each group. Data are presented as means ± standard errors and were analysed using 1-way ANOVA and Dunnett’s multiple comparisons test. *P < 0.05 compared with controls, **P < 0.01 compared with controls. Uncropped western blot images are shown in Supplementary Fig. . Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; SE: status epilepticus; Con: control.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Expressing, Positron Emission Tomography, Injection, Western Blot, Control

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Glucose deficiency influenced TFCP2 and ASIC2a expression in PC12 cells. ( a ) Representative immunoblot and densitometric analyses showing that cells grown in low-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 12 and 24 h of growth. ( b ) Representative immunoblot and densitometric analyses showing that cells grown in no-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 6, 12, and 24 h of growth. ( c ) Representative immunoblot and densitometric analyses showing that STF-31-treated PC12 exhibited significant downregulation of TFCP2 and significant upregulation of ASIC2a relative to DMSO-treated control cells. ( d ) Representative immunoblot and densitometric analyses showing TFCP2 and ASIC2a expression in 2-deoxy-D-glucose-treated PC12 cells cultured in no-glucose media. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using 1-way ANOVA and Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01 compared to controls; # P < 0.05, ## P < 0.01 compared to the DMSO group. Uncropped western blot images are shown in Supplementary Fig. . Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; DMSO: dimethyl sulfoxide; 2-DG: 2-deoxy-D-glucose; Con: control.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Expressing, Western Blot, Control, Cell Culture

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: TFCP2 inversely regulates ASIC2a expression. ( a ) Representative immunoblot and densitometric analyses showing that TFCP2 siRNA-transfected PC12 cells had 27% lower TFCP2 expression than negative siRNA-treated cells and consequently elevated ASIC2a expression. (b ) Representative immunoblot and densitometric analyses showing that TFCP2 OE plasmid-transfected PC12 cells had 1.76 ± 0.45-fold greater TFCP2 expression than negative control cells and consequently suppressed ASIC2a expression. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P < 0.05 compared to negative controls. Uncropped western blot images are shown in Supplementary Fig. . Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; siRNA: short interfering RNA; OE: overexpression; KD: knockdown; NC: negative control.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Negative Control, Small Interfering RNA, Over Expression, Knockdown

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Cellular localisation of TFCP2 and ASIC2a in the epileptic rats’ CA1 regions and glucose-deficient cells. (a ) Double immunofluorescence labelling for TFCP2 and ASIC2a in the epileptic rats’ CA1 regions during acute and latent post-seizure phases. ( b ) Cellular localisation of TFCP2 and ASIC2a expression in PC12 cells grown for 24 h in high-glucose media (control), low-glucose media, no-glucose media, and STF-31 (10 µM)-loaded media. Proteins were probed with anti-ASIC2a (green) and anti-TFCP2 (red) antibodies. Nuclei were counterstained with DAPI (blue). Scale bars = 20 μm. Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Immunofluorescence, Expressing, Control

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Changes in ASIC2a expression affected the intrinsic excitability of CA1 pyramidal neurons. ( a ) Confocal images of CA1 pyramidal neurons expressing GFP (green) and labelled with neurobiotin (blue). Scale bar = 10 μm. ( b ) Representative traces of action potential firing in response to 200 pA current injections in CA1 pyramidal neurons of the negative control, ASIC2a overexpression, and ASIC2a knockdown groups, respectively. ( c ) Number of action potentials in CA1 pyramidal neurons from the various groups at different current injection steps. Data are presented as means ± standard errors and were analysed using 1- or 2-way ANOVA and Dunnett's multiple comparisons test. *P < 0.05, ASIC2a OE group compared with negative control group; # P < 0.05, ASIC2a KD group compared with negative control group. Abbreviations, ASIC2a: acid-sensing ion channel 2a; GFP: green fluorescent protein; OE: overexpression; KD: knockdown.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Expressing, Negative Control, Over Expression, Knockdown, Injection

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Hippocampal ASIC2a overexpression increased seizure susceptibility. ( a ) Representative images showing GFP immunoreactivity in the hippocampal CA1 region following adeno-associated virus (AAV) vector infusion. Scale bar = 100 μm. ( b ) The time interval from pilocarpine injection to Racine IV seizures was significantly shorter in the ASIC2a overexpression group (26.3 ± 1.3 min) than in the negative control AAV group (31.9 ± 1.9) (n = 30 rats/group). ( c ) The proportion of rats with Racine IV seizures after pilocarpine treatment was significantly higher in the ASIC2a overexpression group (93.3 ± 3.33%) than in the negative control AAV group (66.7 ± 3.33%). Data are presented as means ± standard errors and were analysed using unpaired t-tests or Chi-square tests. *P < 0.05 compared with negative control group. Abbreviations, ASIC2a: acid-sensing ion channel 2a; GFP: green fluorescent protein; DAPI: 4′, 6-diamidino-2-phenylindole; OE: overexpression.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Over Expression, Virus, Plasmid Preparation, Injection, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

doi: 10.1111/jcmm.14238

Figure Lengend Snippet: Immunohistochemical analysis of ASIC1a expression in renal tissue. Immunostaining for ASIC1a, AQP1, THP and SYN in renal tissue. ASIC1a was colocalized with AQP and SYN, but not THP. ASIC1a, acid sensing ion channel 1a; AQP1, aquaporin 1, proximal tubular cells marker; THP, Tamm‐Horsfall protein, thick ascending limb and distal tubular cells marker; SYN, Synaptopodin, podocyte marker; DAPI, 4,6‐diamidino‐2‐phenylindole, nuclear

Article Snippet: After incubated in 0.3% BSA, the slices was incubated in the mixed primary antibodies: Guinea pig anti‐ASIC1a (1:50 alomone lab, Israel)+Rabbit anti‐aquaporin 1 (AQP1, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐ Tamm‐Horsfall protein (THP, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐Synaptopodin (SYN, 1:100, Abcam, USA), Guinea pig anti‐ASIC2a (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐SYN (1:100), Guinea pig anti‐ASIC3 (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐ SYN (1:100).

Techniques: Immunohistochemical staining, Expressing, Immunostaining, Marker

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Altered hippocampal TFCP2 and ASIC2a expression with glucose hypometabolism in patients with TLE. ( a ) Patient 4’s pre-surgical assessment results: magnetic resonance imaging (left) was negative, electroencephalography (middle) showed spike waves in the temporal lobe, and fluorodeoxyglucose positron emission tomography (right) revealed hypometabolic lesions in the right hippocampus. ( b ) Representative western blot assays of hippocampal TFCP2 and ASIC2a expression in patients with TLE (n = 13) and control patients (n = 10). β-actin was used as a loading control. ( c , d ) Normalised densitometry bar graphs of TFCP2 and ASIC2a for the control subjects and patients with TLE. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P < 0.05, **P < 0.01 compared to controls. Uncropped western blot images are shown in Supplementary Fig. . Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; TLE: temporal lobe epilepsy.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Expressing, Magnetic Resonance Imaging, Positron Emission Tomography, Western Blot

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Altered hippocampal TFCP2 and ASIC2a expression with glucose hypometabolism in pilocarpine-treated rats. ( a ) Representative coronal view microscopic positron emission tomography images in the different phases of epileptogenesis. ( b ) Hippocampal glucose uptake in the different phases after pilocarpine injection. ( c ) Representative western blot assays of hippocampal TFCP2 and ASIC2a expression at different post-seizure time points. ( d ) Bar graph showing decreased hippocampal TFCP2 expression in pilocarpine-treated rats. ( e ) Bar graph showing increased hippocampal ASIC2a expression in pilocarpine-treated rats. The experiments were repeated at least 3 times with at least 3 rats in each group. Data are presented as means ± standard errors and were analysed using 1-way ANOVA and Dunnett’s multiple comparisons test. *P < 0.05 compared with controls, **P < 0.01 compared with controls. Uncropped western blot images are shown in Supplementary Fig. . Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; SE: status epilepticus; Con: control.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Expressing, Positron Emission Tomography, Injection, Western Blot

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Glucose deficiency influenced TFCP2 and ASIC2a expression in PC12 cells. ( a ) Representative immunoblot and densitometric analyses showing that cells grown in low-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 12 and 24 h of growth. ( b ) Representative immunoblot and densitometric analyses showing that cells grown in no-glucose media had significantly decreased TFCP2 expression and significantly increased ASIC2a expression relative to those grown in high-glucose media after 6, 12, and 24 h of growth. ( c ) Representative immunoblot and densitometric analyses showing that STF-31-treated PC12 exhibited significant downregulation of TFCP2 and significant upregulation of ASIC2a relative to DMSO-treated control cells. ( d ) Representative immunoblot and densitometric analyses showing TFCP2 and ASIC2a expression in 2-deoxy-D-glucose-treated PC12 cells cultured in no-glucose media. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using 1-way ANOVA and Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01 compared to controls; # P < 0.05, ## P < 0.01 compared to the DMSO group. Uncropped western blot images are shown in Supplementary Fig. . Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; DMSO: dimethyl sulfoxide; 2-DG: 2-deoxy-D-glucose; Con: control.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Expressing, Western Blot, Cell Culture

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: TFCP2 inversely regulates ASIC2a expression. ( a ) Representative immunoblot and densitometric analyses showing that TFCP2 siRNA-transfected PC12 cells had 27% lower TFCP2 expression than negative siRNA-treated cells and consequently elevated ASIC2a expression. (b ) Representative immunoblot and densitometric analyses showing that TFCP2 OE plasmid-transfected PC12 cells had 1.76 ± 0.45-fold greater TFCP2 expression than negative control cells and consequently suppressed ASIC2a expression. The experiments were repeated at least 3 times. Data are presented as means ± standard errors and were analysed using unpaired t-tests. *P < 0.05 compared to negative controls. Uncropped western blot images are shown in Supplementary Fig. . Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; siRNA: short interfering RNA; OE: overexpression; KD: knockdown; NC: negative control.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Negative Control, Small Interfering RNA, Over Expression

Journal: Scientific Reports

Article Title: Glucose Deficiency Elevates Acid-Sensing Ion Channel 2a Expression and Increases Seizure Susceptibility in Temporal Lobe Epilepsy

doi: 10.1038/s41598-017-05038-0

Figure Lengend Snippet: Cellular localisation of TFCP2 and ASIC2a in the epileptic rats’ CA1 regions and glucose-deficient cells. (a ) Double immunofluorescence labelling for TFCP2 and ASIC2a in the epileptic rats’ CA1 regions during acute and latent post-seizure phases. ( b ) Cellular localisation of TFCP2 and ASIC2a expression in PC12 cells grown for 24 h in high-glucose media (control), low-glucose media, no-glucose media, and STF-31 (10 µM)-loaded media. Proteins were probed with anti-ASIC2a (green) and anti-TFCP2 (red) antibodies. Nuclei were counterstained with DAPI (blue). Scale bars = 20 μm. Abbreviations, ASIC2a: acid-sensing ion channel 2a; TFCP2: transcription factor CP2; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The sections were then incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-ASIC2a antibody (1:100, Abcam) and mouse monoclonal anti-TFCP2 antibody (1:100, BD Biosciences).

Techniques: Immunofluorescence, Expressing