Journal: Pharmaceuticals

Article Title: Segmental Upregulation of ASIC1 Channels in the Formalin Acute Pain Mouse Model

doi: 10.3390/ph15121539

Figure Lengend Snippet: ASIC1 levels in the pain pathway. ( A ) Representative membrane of lysates of ACC tissue from formalin (For)- or vehicle (Veh)- injected male mice at Ip (ipsilateral) or Con (contralateral) sides to the injection detected using ASIC1 and tubulin antibodies (left), and plot of the results obtained from membranes for ASIC1/tubulin detected levels (right) (4–5 animals used per condition). ( B ) Representative membrane of lysates of SC tissue from For- or Veh- injected mice at different lumbar (3, 4, 5) levels detected using ASIC1 and tubulin antibodies (left), and plot of the results obtained from membranes for ASIC1/tubulin detected levels (right) (4–5 animals used per condition). Notice the gradient decrease from level 3 to 5. ( C ) Representative membrane of lysates of a pool of DRG tissue from different lumbar (3, 4, 5) levels in For- or Veh- injected male mice at Ip or Con sides to the injection detected using ASIC1 and tubulin antibodies. Notice the same gradient pattern as in ( B , D ) Images of the regions dissected (SC, DRG segments) used for the experiments in ( B , C ). One-way ANOVA, **** p < 0.0001; *** p < 0.001 ** p < 0.01. Scale bar 5 mm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti ASIC1 (Alomone ASC-014, 1:1000); mouse monoclonal anti-tubulin (DM1a; Cell signaling #3873, 1:5000); rabbit polyclonal anti-total ERK (Santa Cruz, C9, 1:500); rabbit polyclonal anti phosphoERK (Cell Signaling, SC-7383, 1:500).

Techniques: Injection

Journal: Pharmaceuticals

Article Title: Segmental Upregulation of ASIC1 Channels in the Formalin Acute Pain Mouse Model

doi: 10.3390/ph15121539

Figure Lengend Snippet: Schematic representation of ASIC1 upregulation in ACC, SC and DRG in the formalin mouse model of pain. At the cortex, the ACC contralateral to the injection (left hind paw) shows a higher ASIC1 protein level. At the SC and DRGs, there is a gradient decreasing from L3 to L5 lumbar segments. Spinal nerves contribution to the sciatic nerve is represented with lines, thicker for those contributing to a greater extent. ASIC1 protein levels are represented showing an increase with more intense color. (IP, ipsilateral; CON, contralateral; ACC, Anterior Cingulate Cortex; DRG (Dorsal Root Ganglia; SC, Spinal Cord; L3, 4, 5, lumbar 3, 4, 5).

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti ASIC1 (Alomone ASC-014, 1:1000); mouse monoclonal anti-tubulin (DM1a; Cell signaling #3873, 1:5000); rabbit polyclonal anti-total ERK (Santa Cruz, C9, 1:500); rabbit polyclonal anti phosphoERK (Cell Signaling, SC-7383, 1:500).

Techniques: Injection

Journal: The Journal of Neuroscience

Article Title: Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes

doi: 10.1523/JNEUROSCI.3781-15.2016

Figure Lengend Snippet: Marker expression for T7 DRG neurons in the absence of Merkel cells

Article Snippet: The primary antibodies used were mouse anti-Islet1/2 (1:50; catalog #39.4D5, Developmental Studies Hybridoma Bank), rabbit anti-NF200 (1:1000; N4142, Sigma-Aldrich), goat anti-TrkB (1:200; AF1494, R&D Systems), goat anti-TrkC [AF1404 (1:100) and BAF1404 (1:20), R&D Systems], rabbit anti-Ret (1:50; catalog #18121, Immuno-Biological Laboratories), rabbit anti-parvalbumin (PV; 1:1000; PV25, Swant), rabbit anti-CGRP (1:1000; T-4032, Peninsula Laboratories), rabbit anti-phospho-SMAD1/5/8 (1:250; catalog 9511, Cell Signaling Technology), goat anti-TBX3 (1:100; sc-31656, Santa Cruz Biotechnology), rat anti-Keratin8 (1:20, TROMA-1, Developmental Studies Hybridoma Bank), guinea pig anti-Asic1 (1:250; Alomone Labs), rabbit anti-δ opioid receptor (DOR; 1:500; Alomone Labs), and rabbit anti-γENaC (1:500; StressMarq Biosciences).

Techniques: Marker, Expressing

Journal: The Journal of Neuroscience

Article Title: Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes

doi: 10.1523/JNEUROSCI.3781-15.2016

Figure Lengend Snippet: Merkel cell deletion alters the expression of SAI ion channel components. A, qPCR analysis of ion channels in P21 thoracic DRGs (n ≥ 8 mice/genotype at each age). B–G, Immunostaining for NF200, γENaC, Asic1, and DOR in T7 DRG sections from P21 control littermate and K14; Atoh1CKO mice. Asterisks indicate double-positive cells. H, Percentages of Islet1/2+ T7 DRG neurons that were NF200+ γENaC+, NF200+Asic1, and NF200+DOR+ in P21 control and K14; Atoh1CKO mice (n ≥ 3 mice/genotype). Error bars in graphs represent the SEM, and asterisks indicate statistically significant differences between genotypes. *p < 0.05, ***p < 0.001.

Article Snippet: The primary antibodies used were mouse anti-Islet1/2 (1:50; catalog #39.4D5, Developmental Studies Hybridoma Bank), rabbit anti-NF200 (1:1000; N4142, Sigma-Aldrich), goat anti-TrkB (1:200; AF1494, R&D Systems), goat anti-TrkC [AF1404 (1:100) and BAF1404 (1:20), R&D Systems], rabbit anti-Ret (1:50; catalog #18121, Immuno-Biological Laboratories), rabbit anti-parvalbumin (PV; 1:1000; PV25, Swant), rabbit anti-CGRP (1:1000; T-4032, Peninsula Laboratories), rabbit anti-phospho-SMAD1/5/8 (1:250; catalog 9511, Cell Signaling Technology), goat anti-TBX3 (1:100; sc-31656, Santa Cruz Biotechnology), rat anti-Keratin8 (1:20, TROMA-1, Developmental Studies Hybridoma Bank), guinea pig anti-Asic1 (1:250; Alomone Labs), rabbit anti-δ opioid receptor (DOR; 1:500; Alomone Labs), and rabbit anti-γENaC (1:500; StressMarq Biosciences).

Techniques: Expressing, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: Relative mRNA levels from D54-MG glioblastoma cells and primary human astrocytes. This figure shows average relative mRNA expression for different ENaC/Deg subunits proportional to 18 S in D54-MG glioblastoma cells and primary human astrocytes. A significantly higher expression for ASIC1 (A), αENaC (B), and γENaC (C) is seen in D54-MG cells compared with astrocytes. The results are the averages of six different samples of D54-MG, and astrocytes ran in duplicate with similar results. Error bars ± 1 S.D. The asterisk indicates p < 0.05.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: Knockdown of αENaC inhibits the basal whole cell current seen in D54-MG cells. A, diagram depicting the schematic of the αENaC dominant negative constructs. B1, immunoblot showing reduced expression of αENaC protein in DN-αENaC cDNA-transfected D54-MG cells compared with untransfected D54-MG control cells. B2, quantification of the blots show a 60% reduction of αENaC protein in transfected cells compared with untransfected cells. To look for the specificity of αENaC knockdown, cell lysates from B were probed for expression of ASIC1 (C1 and C2), and no difference was found in ASIC1 expression in D54-MG cells after αENaC knockdown compared with untransfected cells; n = 3 for all the blots. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. D, representative traces of untransfected D54-MG cells before and after infusion of 100 μm amiloride (top panel) and after knocking down αENaC (lower panel). E, the corresponding I/V curve and average conductance at −100 mV (F) showing a significant inhibition of inward current after the addition of 100 μm amiloride or after knocking down αENaC. Control traces are the same as in Fig. 3; for αENaC knockdown, n = 5. Error bars are ±1 S.D. The asterisk indicates p < 0.05.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Dominant Negative Mutation, Construct, Western Blot, Expressing, Transfection, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: Knockdown of ASIC1 cDNA and whole cell patch clamp recording. A, diagram depicting the schematic of the ASIC1 dominant negative constructs. Representative Western blot (B1) of lysates from D54-MG glioma cells transfected with DN eGFP-ASIC1 cDNA or untransfected control D54-MG cells probed for ASIC1 and quantified (B2) showing a 50–60% inhibition in ASIC1 protein expression in ASIC1 knockdown D54-MG cells compared with untransfected cells. C1, cell lysates from B were used to look for the specificity of dominant negative mutation techniques in knocking down the protein of interest. Lysates were probed by Western blot for γENaC protein expression. No difference was found in the γENaC protein expression between ASIC1 knockdown and control D54-MG cells (C2). Total actin (B1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; C1) were used as the loading control; n = 3 for all the blots. D, representative whole-cell patch clamp records of control cells (upper panel) and those transfected with ASIC1 dominant negative cDNA for 48 h (lower panel). E, I/V curve showing significant inhibition of whole cell current after the addition of 100 μm amiloride to the bath solution or after knocking down ASIC1 in D54-MG glioma cells. F, average conductance at −100 mV for each condition as in B showing significant inhibition of inward current after the addition of 100 μm amiloride or after knocking down ASIC. Traces for D54 control n = 14, for D54 + 100 μm amiloride n = 10, for ASIC1 DN and ASIC1 DN + 100 μm amiloride n = 4. Error bars ±1 S.D. The asterisk indicates p < 0.05.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Patch Clamp, Dominant Negative Mutation, Construct, Western Blot, Transfection, Inhibition, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: γENaC Knockdown and whole cell patch clamp recording in D54-MG glioma cells. A, diagram depicting the schematic of the γENaC dominant negative constructs. B1, immunoblot showing reduced expression of γENaC protein in DN-γENaC cDNA-transfected D54-MG cells compared with untransfected D54-MG control cells. B2, quantification of the blots showing a 60–70% reduction of γENaC protein in transfected cells compared with untransfected cells. To look for the specificity of γENaC knockdown, cell lysates from B were probed for expression of ASIC1 (C1 and C2) and αENaC (D1 and D2). Immunoblots showed similar expression of ASIC1 and αENaC in both DN-γENaC cDNA-transfected D54-MG cells and in control untransfected cells. Actin was used as loading control in all traces; n = 3 for all the blots. E, representative traces of untransfected D54-MG cells before and after infusion of 100 μm amiloride (top panel) and after knocking down γENaC (lower panel). The corresponding I/V curve (F) and average conductance at −100 mV (G) showing a significant inhibition of inward current after the addition of 100 μm amiloride or after knocking down γENaC. Control traces are the same as in Fig. 3; for γENaC knockdown, n = 6. Error bars ±1 S.D. The asterisk indicates p < 0.05.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Patch Clamp, Dominant Negative Mutation, Construct, Western Blot, Expressing, Transfection, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: ENaC/Deg subunits interact with each other in glioma cells. A, lysate from CHO-K1 cells transfected with GFP ligated ASIC1 cDNA was immunoprecipitated (IP) with mouse anti-GFP or rabbit anti-ASIC1 antibody and blotted with mouse anti-GFP antibody. The Western blot shows that immunoprecipitating with either mouse anti-GFP or rabbit anti-ASIC1 antibody and blotting with mouse anti-GFP antibody pulled down GFP-ASIC1 at 100 kDa. Western blots of total membrane fractions (B) and plasma membrane fractions (C) isolated from D54-MG cells stably transfected with ASIC1-GFP and immunoprecipitated with mouse anti-GFP, rabbit anti-ASIC1, rabbit anti-αENaC, or rabbit anti-γENaC antibody show an interaction of ASIC1 with αENaC and γENaC in D54-MG cells. Non-immune mouse IgG immunoprecipitation and immunoprecipitation with only protein A-agarose beads showed that the anti-GFP antibody was specific. D, to rule out that ASIC1 and ENaC subunit interactions were because of a random association due to overexpression, CHO-K1 and D54-MG cells were transfected with an unrelated protein; CFP ligated CLC1. Lysates from both CHO-K1 and D54-MG cells immunoprecipitated (IP) with mouse anti-GFP antibody pulled down CFP-CLC1 upon blotting with mouse anti-GFP antibody, whereas immunoprecipitating D54-MG cell lysate with rabbit anti-ASIC1 antibody did not pull down CLC1 upon blotting with mouse anti-GFP antibody. n = 3 for all the blots.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Transfection, Immunoprecipitation, Western Blot, Isolation, Stable Transfection, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

doi: 10.1074/jbc.M109.037390

Figure Lengend Snippet: Knocking down ASIC1, αENaC, or γENaC inhibits D54-MG glioma cell migration. Representative images are shown of migrated D54-MG cells transfected with eYFP (A), with 100 μm benzamil in the migration buffer (B), with ASIC1 knockdown (C), αENaC knockdown (D), or with γENaC knockdown (E). The average number of cells that had migrated per field under each conditions (F) is shown. Error bars are ±1 S.D., and the asterisk indicates p < 0.05 comparing cell migration for D54-YFP with cell migration under other conditions. n = 3 for each experiment except D54-YFP, where n = 6.

Article Snippet: Real time PCR to measure ASIC1 (Hs00241630_m1), αENaC (Hs00168906_m1), γENaC (Hs00168918_m1), and δENaC (Hs00161595_m1) mRNA was performed using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, catalog no. 4309169) using the manufacturer's handbook as a reference (Relative Quantification; Applied Biosystems 7300/7500 Real Time PCR system; 2004).

Techniques: Migration, Transfection