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