rabbit anti nalcn  (Alomone Labs)

Journal: Frontiers in Molecular Neuroscience

Article Title: Ectopic expression of Nav1.7 in spinal dorsal horn neurons induced by NGF contributes to neuropathic pain in a mouse spinal cord injury model

doi: 10.3389/fnmol.2023.1091096

Figure Lengend Snippet: Expression of voltage-gated sodium channels in spinal dorsal horn and DRG of SCI mice at 12 DPO. (A) Western blot showed that the expression level of Nav1.2 was not significantly upregulated in SDH of SCI mice when compared to that in naive mice. (B) Western blot demonstrated that there was no significant difference in the expression level of Nav1.8 in SDH between SCI mice and naive mice. (C) The expression level of Nav1.7 in SDH of naive and SCI mice measured by Western-blot assay. (D) The expression level of Nav1.7 in DRG significantly increased 1.5-fold (1.5 ± 0.32) in SCI mice compared to that in naive mice. Data are shown as Mean ± SD, * p < 0.05, n = 4, unpaired t-test . n.s. not significant. Scale bar = 100 μm.

Article Snippet: Primary antibodies included: rabbit antibodies against Nav1.2 (Alomone labs, ASC-022, 1:100), NGF (Abcam, ab52918, 1:1,000), Phospho-c-JUN (Cell signaling technology, #9261, 1:1,000), and GAPDH (Proteintech, 60004-1-Ig, 1:3,000), and mouse antibodies against Nav1.7 (Abcam, ab85015, 1:800) and Nav1.8 (NeuroMab, 75–166, USA, 1:1,000).

Techniques: Expressing, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Ectopic expression of Nav1.7 in spinal dorsal horn neurons induced by NGF contributes to neuropathic pain in a mouse spinal cord injury model

doi: 10.3389/fnmol.2023.1091096

Figure Lengend Snippet: Upregulation of Nav1.7 in DRG and SDH of SCI mice. (A) The expression level of Nav1.7 in DRG and SDH of naive, sham, and SCI mice was measured by Western blot. (B) Quantitative analysis of duplicate Western-blot membranes showed that compared to naive mice, the expression level of Nav1.7 increased significantly in DRG of SCI mice (1.3 ± 0.10), and an increasing trend in expression level of Nav1.7 was also observed in sham mice (1.1 ± 0.19). The expression level of Nav1.7 in SDH of SCI and sham mice was 2.2-fold and 1.5-fold higher than that in naive mice, respectively, (2.2 ± 1.50, 1.5 ± 1.03). (C) Immunostaining for Nav1.7 (Green) on spinal section derived from naive, sham, and SCI mice. Arrows point to Nav1.7 positive neurons, DAPI is counterstaining for nuclei. Inset is the high magnification view of the boxed area. (D) The number of neurons expressing Nav1.7 in laminae I-VI of naive, sham, and SCI mice. # represents the comparison between SCI and sham，* represents the comparison between naïve and SCI or sham. (E) Immunostaining for Nav1.7 (Green) on DRG section derived from naive, sham, and SCI mice demonstrated the upregulation of Nav1.7 in DRG neurons in SCI and sham mice when compared to that in naive mice. Inset is the high magnification view of the boxed area. Data are shown as Mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001, unpaired t-test or one way ANOVA. n.s. not significant. Scale bar = 100 μm.

Article Snippet: Primary antibodies included: rabbit antibodies against Nav1.2 (Alomone labs, ASC-022, 1:100), NGF (Abcam, ab52918, 1:1,000), Phospho-c-JUN (Cell signaling technology, #9261, 1:1,000), and GAPDH (Proteintech, 60004-1-Ig, 1:3,000), and mouse antibodies against Nav1.7 (Abcam, ab85015, 1:800) and Nav1.8 (NeuroMab, 75–166, USA, 1:1,000).

Techniques: Expressing, Western Blot, Immunostaining, Derivative Assay, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Ectopic expression of Nav1.7 in spinal dorsal horn neurons induced by NGF contributes to neuropathic pain in a mouse spinal cord injury model

doi: 10.3389/fnmol.2023.1091096

Figure Lengend Snippet: Blockers of Nav1.7-alleviated mechanical pain in SCI mice. (A) Nav1.7 blocker PF-05089771 significantly relieved mechanical pain of SCI mice at doses of both 2 mg/kg and 4 mg/kg (Vehicle 0.03 ± 0.08 g, 2 mg/kg 30 min 0.2 ± 0.13 g, 4 mg/kg 30 min 0.3 ± 0.16 g, 2 mg/kg 60 min 0.4 ± 0.19 g, and 4 mg/kg 60 min 0.4 ± 0.20) g. △PWT = post-drug PWT - pre-drug PWT. (B) Nav1.7 blocker GNE-0439 significantly alleviated mechanical pain of SCI mice at doses from 10 μg/kg to 30 μg/kg (10 μg/kg 30 min 0.3 ± 0.15 g, 20 μg/kg 30 min 0.5 ± 0.28 g, 30 μg/kg 30 min 0.2 ± 0.11 g, 10 μg/kg 60 min 0.2 ± 0.18 g, 20 μg/kg 60 min 0.3 ± 0.20 g, and 30 μg/kg 60 min 0.2 ± 0.08 g). (C) Gabapentin significantly reduced mechanical pain of SCI mice at dose of 50 mg/kg (30 min 0.5 ± 0.36 g and 60 min 0.5 ± 0.30 g). (D) The efficacy of GNE-0439 to relieve mechanical pain was equivalent to Gabapentin, and the efficacy of GNE-0439 and Gabapentin was slightly better than PF-05089771, but not statistically significant (PF-05089771 0.4 ± 0.20 g, GNE-0439 0.5 ± 0.28 g, and Gabapentin 0.5 ± 0.36 g). (E) Response ratio of paw withdrawal of SCI mice administrated neither PF-05089771 (blue line) or GNE-0439 (red line) to stimulus from 0.07 g Von Frey monofilament to 1.0 g Von Frey monofilament. Data are shown as Mean ± SD, n = 7. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired t-test or one-way ANOVA, or two-way ANOVA. n.s. not significant.

Article Snippet: Primary antibodies included: rabbit antibodies against Nav1.2 (Alomone labs, ASC-022, 1:100), NGF (Abcam, ab52918, 1:1,000), Phospho-c-JUN (Cell signaling technology, #9261, 1:1,000), and GAPDH (Proteintech, 60004-1-Ig, 1:3,000), and mouse antibodies against Nav1.7 (Abcam, ab85015, 1:800) and Nav1.8 (NeuroMab, 75–166, USA, 1:1,000).

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Ectopic expression of Nav1.7 in spinal dorsal horn neurons induced by NGF contributes to neuropathic pain in a mouse spinal cord injury model

doi: 10.3389/fnmol.2023.1091096

Figure Lengend Snippet: Activation of Nav1.7 + SDH neurons in SCI mice. (A–L) Double immunostaining for FOS (Red) and Nav1.7 (Green) on spinal section derived from naive (A, D, G, J) , sham (B, E, H, K) , and SCI (C, F, I, L) mice showed more FOS-positive neurons and more FOS/Nav1.7 double-positive neurons in deep laminae layers of SCI mice. Inset is the high magnification view of the boxed area. Scale bar 100 μm. (M–O) The number of FOS + (M) , Nav1.7 + /FOS + (N) , and Nav1.7 + (O) SDH neurons in laminae I–VI of home-caged naive mice, sham, and SCI mice. The symbol # represents the comparison between SCI and sham, and the symbol * represents the comparison between naive and SCI or sham. (P) The ratio of Nav1.7 + /FOS + SDH neurons to total Nav1.7 + SDH neurons in naive (0%), sham (3 ± 4.8%), and SCI (26 ± 8.2%) mice. Data are shown as Mean ± SD, n = 3. # p < 0.05, ## p < 0.01,* p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t -test or one way ANOVA. Scale bar = 100 μm.

Article Snippet: Primary antibodies included: rabbit antibodies against Nav1.2 (Alomone labs, ASC-022, 1:100), NGF (Abcam, ab52918, 1:1,000), Phospho-c-JUN (Cell signaling technology, #9261, 1:1,000), and GAPDH (Proteintech, 60004-1-Ig, 1:3,000), and mouse antibodies against Nav1.7 (Abcam, ab85015, 1:800) and Nav1.8 (NeuroMab, 75–166, USA, 1:1,000).

Techniques: Activation Assay, Double Immunostaining, Derivative Assay, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Ectopic expression of Nav1.7 in spinal dorsal horn neurons induced by NGF contributes to neuropathic pain in a mouse spinal cord injury model

doi: 10.3389/fnmol.2023.1091096

Figure Lengend Snippet: Schematic view of mechanism underlying SCI-induced NP. (A) . SCI induced the upregulation of NGF, and consequently increased phosphorylated JUN and the upregulation of Nav1.7 in SDH and DRG neurons of mice. (B) . Nav1.7 selective blockers attenuated SCI-induced NP in mice through inhibiting the activity of Nav1.7 in both the spinal cord and DRG.

Article Snippet: Primary antibodies included: rabbit antibodies against Nav1.2 (Alomone labs, ASC-022, 1:100), NGF (Abcam, ab52918, 1:1,000), Phospho-c-JUN (Cell signaling technology, #9261, 1:1,000), and GAPDH (Proteintech, 60004-1-Ig, 1:3,000), and mouse antibodies against Nav1.7 (Abcam, ab85015, 1:800) and Nav1.8 (NeuroMab, 75–166, USA, 1:1,000).

Techniques: Activity Assay