Journal: Neurobiology of Pain

Article Title: Role of voltage-gated sodium channels in axonal signal propagation of trigeminal ganglion neurons after infraorbital nerve entrapment

doi: 10.1016/j.ynpai.2022.100084

Figure Lengend Snippet: Increased sensitivity of infraorbital nerve to axonal conduction block by tetrodotoxin (TTX) and NaV1.8 blocker after IoNE. A-B: Sample recording traces from an IoNE rat show decreases in A-fiber CAP (10 mA; 0.1 ms) area and C-fiber CAP (15 mA; 1 ms) amplitudes after TTX and NaV1.8 blocker, A-803467 (A8). C-D: Decreases in A-fiber CAP area and C-fiber CAP amplitude are significantly greater in IoNE compared to Sham after bath application of TTX (300 nM) and TTX (300 nM) + A-803467 (5 μM). However, the decrease in A- and C-fiber CAP measurements of IoNE group are not significantly different from Sham after TTX (100 nM). *, p < 0.05; **, p < 0.01 Sham vs IoNE; two-way ANOVA, Sidak’s multiple comparison test (n = 7–8/group).

Article Snippet: Primary antibodies for target proteins were diluted in Wes antibody diluent 2 as follows: NaV1.3 (1:20), NaV1.6 (1:20), NaV1.7 (1: 150), NaV1.8 (1:100), and NaV1.9 (1:20) (Alomone Labs, Jerusalem, Israel).

Techniques: Blocking Assay

Journal: Neurobiology of Pain

Article Title: Role of voltage-gated sodium channels in axonal signal propagation of trigeminal ganglion neurons after infraorbital nerve entrapment

doi: 10.1016/j.ynpai.2022.100084

Figure Lengend Snippet: Infraorbital nerve entrapment (IoNE) selectively increases the axonal signal propagation of Aδ-fibers innervating the vibrissal pad. A. Schematic of recording arrangement showing the peripheral end of vibrissal pad afferents placed into a stimulating suction electrode and the compound action potentials (CAP) recorded from the proximal end of the nerve with a recording suction electrode. The signals from the artifact suppression and recording electrodes were fed into a differential recording amplifier, digitized, and stored for off-line analysis. B. Sample recording traces show CAP areas (indicated by dotted lines) of Aβ- and Aδ-fibers of IoNE and Sham nerves acquired at 10 mA stimulus strength, 0.1 ms duration. The vertical dotted lines indicate an approximate conduction velocity of 8.5 m/s, used to segregate Aβ- and Aδ-fiber CAP areas C-D: Stimulus-output response plots show no significant difference in CAP areas of Aβ-fibers between IoNE and Sham nerves. In contrast, IoNE significantly increased the Aδ-fiber CAP area of infraorbital nerve samples at higher stimulus strengths compared to Sham nerves. E-F: Changes in Aβ- and Aδ-fiber CAP areas expressed as percent of vehicle control (DMSO) after cumulative application of NaV1.7 blocker (ICA-121431, 1 μM), NaV1.8 blocker (A-803479, 5 μM), 4,9-anhydro-TTX (0.5 μM), and TTX (100 nM). *, p < 0.05; **, p < 0.01; ***, p < 0.001 Sham vs IoNE; two-way ANOVA, Sidak’s multiple comparison test (n = 5–8/group).

Article Snippet: Primary antibodies for target proteins were diluted in Wes antibody diluent 2 as follows: NaV1.3 (1:20), NaV1.6 (1:20), NaV1.7 (1: 150), NaV1.8 (1:100), and NaV1.9 (1:20) (Alomone Labs, Jerusalem, Israel).

Techniques:

Journal: Neurobiology of Pain

Article Title: Role of voltage-gated sodium channels in axonal signal propagation of trigeminal ganglion neurons after infraorbital nerve entrapment

doi: 10.1016/j.ynpai.2022.100084

Figure Lengend Snippet: Changes in the expression of voltage gated sodium channel (NaV) mRNAs in the ipsilateral infraorbital nerves and trigeminal ganglia of Sham and infraorbital nerve entrapment (IoNE) rats measured at 2 weeks post-surgery. A. IoNE selectively increased NaV1.3, NaV1.7, and NaV1.8 mRNAs in the infraorbital nerve, expressed as fold change vs. sham. B. However, in the trigeminal ganglion, IoNE significantly reduced the mRNA levels of NaV1.1, NaV1.5, NaV1.6, NaV1.8, and NaV1.9. Expression of all NaV mRNAs were normalized to the loading control, GAPDH mRNA and to the mean of Sham group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 Sham vs IoNE, two-way ANOVA (n = 8 rats/group).

Article Snippet: Primary antibodies for target proteins were diluted in Wes antibody diluent 2 as follows: NaV1.3 (1:20), NaV1.6 (1:20), NaV1.7 (1: 150), NaV1.8 (1:100), and NaV1.9 (1:20) (Alomone Labs, Jerusalem, Israel).

Techniques: Expressing

Journal: Neurobiology of Pain

Article Title: Role of voltage-gated sodium channels in axonal signal propagation of trigeminal ganglion neurons after infraorbital nerve entrapment

doi: 10.1016/j.ynpai.2022.100084

Figure Lengend Snippet: Quantitative protein analysis of the plasma membrane fraction of ipsilateral infraorbital nerve and trigeminal ganglion samples showing voltage gated sodium channel (NaV) subtype expression in Sham and infraorbital nerve entrapment (IoNE) rats measured at ∼2 weeks post-surgery. A-J. Spectral graphs on left show group averages (n = 8/group) of target peaks: NaV1.3 (A, F); NaV1.6 (B, G); NaV1.7 (C, H); NaV1.8 (D, I); NaV1.9 (E, J). Bar graphs on the right are group averages of target proteins normalized to total protein and to the mean of the Sham group. *, p < 0.05; **, p < 0.01 Sham vs IoNE, unpaired t -test. Note: The plasma membrane expression of NaV1.8 is significantly increased in the infraorbital nerve of IoNE rats compared to sham rats.

Article Snippet: Primary antibodies for target proteins were diluted in Wes antibody diluent 2 as follows: NaV1.3 (1:20), NaV1.6 (1:20), NaV1.7 (1: 150), NaV1.8 (1:100), and NaV1.9 (1:20) (Alomone Labs, Jerusalem, Israel).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Dissection of the Crosstalk between Na V and Ca V Channels in GH3b6 Cells

doi: 10.3390/ijms23020827

Figure Lengend Snippet: Na V channel expression in GH3b6 cells. ( A ) The mRNA expression levels of all α and β subunits were first determined by relative RT-qPCR. ( B ) RT-qPCR with absolute quantification for the genes, which were detected by relative RT-qPCR. One-way ANOVA (**** p < 0.0001) followed by Tukey post-hoc multiple comparison test was performed. The data are mean ± SEM. D: Disregarded (Ct > 32); ND: Not Detected. ( C ) Western blot analysis of Na V channel expression. Immunoblotting was performed using pan-Na V and Nav1.3 channels antibodies with 20 and 50 µg of protein extracts from GH3b6 cells and rat brain, after separation on 8% SDS-PAGE. Actin was the loading control. ( D ) Immunocytolocalization of Na V 1.3 channels in GH3b6 cells. Fluorescence labeling (green) using Alexa Fluor 488 anti-mouse secondary antibody allowed the detection of Na V 1.3 channels at the plasma membrane. Nuclei (blue) were stained with DAPI. Original magnification ×60.

Article Snippet: The primary antibodies used were anti-pan Na V (rabbit, SP19, ASC-003, Alomone labs, Jerusalem, Israel), anti-Na V 1.3 (rabbit, ASC-004, Alomone labs or mouse, WH0006328M1, Sigma-Aldrich Merck), and anti-actin used as loading control (mouse, clones AC-203 or AC-74, Sigma-Aldrich Merck).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, SDS Page, Fluorescence, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Dissection of the Crosstalk between Na V and Ca V Channels in GH3b6 Cells

doi: 10.3390/ijms23020827

Figure Lengend Snippet: Effects of ICA-121431, a selective inhibitor of Na V 1.3 channels, on I Na recorded in GH3b6 cells by manual patch-clamp. ( A , B ) To illustrate the effects of ICA-121431, examples of superimposed I Na elicited by 500 ms depolarizing pulses (at −20, 0, and +20 mV) and examples of superimposed I Na elicited by depolarizing pulse at 0 mV immediately after a 500 ms prepulse (−120, −60, and −50 mV) are shown. The control traces are in blue (CTRL) and red traces correspond to I Na after ICA-121431 application (1 µM). ( B ) Current–voltage relationships and ( C ) activation/inactivation curves of I Na recorded before (blue circles) and after 1 µM ICA-121431 (red circles). Comparison of V 1/2 activation ( D ) and inactivation ( E ) in the absence (CTRL) and in the presence of 1 nM Tf2. Statistical analysis was performed using the two-tailed unpaired t -test, **** p < 0.0001. Data represent the mean ± SEM.

Article Snippet: The primary antibodies used were anti-pan Na V (rabbit, SP19, ASC-003, Alomone labs, Jerusalem, Israel), anti-Na V 1.3 (rabbit, ASC-004, Alomone labs or mouse, WH0006328M1, Sigma-Aldrich Merck), and anti-actin used as loading control (mouse, clones AC-203 or AC-74, Sigma-Aldrich Merck).

Techniques: Patch Clamp, Activation Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Dissection of the Crosstalk between Na V and Ca V Channels in GH3b6 Cells

doi: 10.3390/ijms23020827

Figure Lengend Snippet: Effects of Tf2, a selective β-scorpion toxin of Na V 1.3 channels, on I Na recorded in GH3b6 cells by automated patch-clamp. ( A ) Representative examples of I Na elicited by 50 ms depolarization steps (protocol inset), in the absence and presence of 1 nM Tf2. To illustrate the strong effect of 1 nM Tf2, superimposed I Na traces at −45 mV are shown. ( B ) Current–voltage relationships ( C ) and activation/inactivation curves of I Na recorded before (blue circles) and after 1 nM Tf2 (red circles). Comparison of V 1/2 activation ( D ) and inactivation ( E ) in the absence (CTRL) and in the presence of 1 nM Tf2. Statistical analysis was performed using the two-tailed unpaired t -test, **** p < 0.0001. Data represent the mean ± SEM.

Article Snippet: The primary antibodies used were anti-pan Na V (rabbit, SP19, ASC-003, Alomone labs, Jerusalem, Israel), anti-Na V 1.3 (rabbit, ASC-004, Alomone labs or mouse, WH0006328M1, Sigma-Aldrich Merck), and anti-actin used as loading control (mouse, clones AC-203 or AC-74, Sigma-Aldrich Merck).

Techniques: Patch Clamp, Activation Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Dissection of the Crosstalk between Na V and Ca V Channels in GH3b6 Cells

doi: 10.3390/ijms23020827

Figure Lengend Snippet: Effect of Tf2, a selective β-scorpion toxin of Na V 1.3, on the intracellular Ca 2+ level in GH3b6 cells. ( A ) Representative kinetics of Fura-2 fluorescence emission in GH3b6 cells treated with increasing concentrations of Tf2. ( B ) The Ca 2+ responses induced by Tf2 are concentration dependent. The concentration–response relationships were analyzed using the Hill–Langmuir equation with variable slope. The values of EC 50 and the Hill coefficient were 17.51 ± 1.4 nM and 1.378 (R 2 = 0.99). ( C ) TTX inhibits in a concentration-dependent manner the Ca 2+ responses elicited by 0.5 µM of Tf2 with an IC 50 value of 7.9 ± 2.1 nM and a Hill coefficient of 1.31 ± 0.135, R 2 = 0.94. The data represent the mean ± SEM ( n = 3 wells) and are representative of at least two independent experiments.

Article Snippet: The primary antibodies used were anti-pan Na V (rabbit, SP19, ASC-003, Alomone labs, Jerusalem, Israel), anti-Na V 1.3 (rabbit, ASC-004, Alomone labs or mouse, WH0006328M1, Sigma-Aldrich Merck), and anti-actin used as loading control (mouse, clones AC-203 or AC-74, Sigma-Aldrich Merck).

Techniques: Fluorescence, Concentration Assay

Journal: bioRxiv

Article Title: Lineage transcription factors co-regulate subtype-specific genes providing a roadmap for systematic identification of small cell lung cancer vulnerabilities

doi: 10.1101/2020.08.13.249029

Figure Lengend Snippet: (A) RNA-seq (log2 FPKM) showing expression of SCN3A and KCNB2 , in SCLC tumors (T) or cell lines (C) in the specified SCLC subtype or NSCLC. The line indicates the mean, and the asterisks indicate p-values for each sample compared to SCLC-A tumors. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. (B) UCSC Genome Browser tracks showing ASCL1, NKX2-1, PROX1 and H3K27ac ChIP-seq signals in NCI-H2107 cells around the genes encoding SCN3A and KCNB2 . Black bars above the tracks indicate computationally called binding sites. (C-E) Immunoblots showing SCN3A and KCNB2 protein in NCI-H2107 cells, 72 hours post-transfection with control siRNAs or siRNA targeted against either ASCL1, NKX2-1 , or PROX1 (C), or SCN3A (D) or KCNB2 (E). (F) WST-1 assay for cell viability from cells from (D,E). Each data point represents a biological replicate, and error bars indicate SEM. ANOVA with Bonferroni’s multiple comparisons test was used to determine significant differences relative to control. ns; not significant. (G) Quantification of colony formation assays in soft agar using varying doses of KCNB2 inhibitor Quinine shows increased sensitivity of SCLC-A NCI-H889, compared to SCLC-N NCI-H524, and SCLC-P NCI-H526. (H) Histograms showing SCN3A+ cells are detected in live SCLC-A but not SCLC-N or SCLC-P cell cultures using an SCN3A extracellular domain specific antibody (red). The background fluorescence with the secondary antibody only is shown (gray). (I,J) RT-qPCR for SCN3A (I) and ASCL1 (J) mRNA from FACs isolated SCN3A+ (red) or SCN3A-(black) cells from mixtures of SCLC-A with SCLC-P or – N. Each data point represents a biological sample, error bars = SD around mean, unpaired t-test, * p<0.05, ** p<0.01.

Article Snippet: Cells were incubated with SCN3A antibody (Alomone Labs, Anti-SCN3A (Nav1.3) (extracellular), Cat #: ASC-023, 4.25 µg/ml) for 30 min on ice, washed 3 times with ice cold FACs buffer, incubated with Alexa Fluor 568-conjugated secondary antibody (Thermofisher, Catalog # A10042, 1:400) for 30 min on ice, washed 3 times as above, and resuspended in ice cold FACs buffer.

Techniques: RNA Sequencing Assay, Expressing, ChIP-sequencing, Binding Assay, Western Blot, Transfection, WST-1 Assay, Fluorescence, Quantitative RT-PCR, Isolation