Journal: Journal of Neuroinflammation

Article Title: Vagus nerve stimulation modulates hippocampal inflammation caused by continuous stress in rats

doi: 10.1186/s12974-022-02396-z

Figure Lengend Snippet: aVNS upregulates the expression of α7 nAChR and ChAT in the hippocampus of CS animals. A RT-PCR analysis of α7 nAChR mRNA expression in the hippocampus. Upper images of agarose gel electrophoresis are the representatives of four independent experiments. Quantification of DNA band intensity relative to actin is plotted in lower panel. B Western blot analysis of α7 nAChR in the hippocampal tissues. C Immunofluorescence images of α7 nAChR signals in the granule cell layer (GCL) of the dentate gyrus (DG) and CA3 and CA1 pyramidal cell layers. The nuclei of neurons were visualized by staining with Hoechst 33258 (blue). D Western blot analysis of ChAT protein in the hippocampal tissue. In B and D , upper images are the representatives from four independent experiments. Quantification of band intensity relative to actin is plotted (lower panel). ** p < 0.01, *** p < 0.001 (One-way ANOVA)

Article Snippet: Immunoblotting was performed using primary antibodies including anti-TNF-α (Rabbit-polyclonal, 1:2000; Abcam, Cambridge, UK), anti-IL-1β (Rabbit-polyclonal, 1:2000; Abcam), anti-IL-6 (Mouse-monoclonal, 1:2000; Abcam), anti-α7 nicotinic acetylcholine receptor (Rabbit-polyclonal, 1:200, Alomone Labs, Jerusalem, Israel), anti-choline acetyltransferase (Rabbit-monoclonal, 1:2000; Abcam), anti-5HT1A receptor (Rabbit-polyclonal, 1:1000, Abcam), anti-Iba-1 (Mouse-monoclonal, 1:1000; Fujifilm, Minato, Japan), anti-cleaved Caspase-3 (Rabbit-polyclonal, 1:1000; Cell Signaling Technology, Beverly, MA, USA) and anti-β-actin (Mouse-monoclonal, 1:50,000; Sigma-Aldrich) antibodies.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: Vagus nerve stimulation modulates hippocampal inflammation caused by continuous stress in rats

doi: 10.1186/s12974-022-02396-z

Figure Lengend Snippet: Effects of α7 nAChR inhibitors on the hippocampal production of inflammatory cytokines. An α7 nAChR inhibitor MLA or saline vehicle solution (Veh) was administered 20 min prior to aVNS in CS animals, and proteins were extracted from hippocampal tissues for western blot analysis. Upper images in A – C are the representatives of 4–5 independent experiments for IL-1β, IL-6, and TNF-α proteins. Quantification of protein band intensity relative to actin is plotted in lower panels. ** p < 0.01, *** p < 0.001 (One-way ANOVA)

Article Snippet: Immunoblotting was performed using primary antibodies including anti-TNF-α (Rabbit-polyclonal, 1:2000; Abcam, Cambridge, UK), anti-IL-1β (Rabbit-polyclonal, 1:2000; Abcam), anti-IL-6 (Mouse-monoclonal, 1:2000; Abcam), anti-α7 nicotinic acetylcholine receptor (Rabbit-polyclonal, 1:200, Alomone Labs, Jerusalem, Israel), anti-choline acetyltransferase (Rabbit-monoclonal, 1:2000; Abcam), anti-5HT1A receptor (Rabbit-polyclonal, 1:1000, Abcam), anti-Iba-1 (Mouse-monoclonal, 1:1000; Fujifilm, Minato, Japan), anti-cleaved Caspase-3 (Rabbit-polyclonal, 1:1000; Cell Signaling Technology, Beverly, MA, USA) and anti-β-actin (Mouse-monoclonal, 1:50,000; Sigma-Aldrich) antibodies.

Techniques: Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: NBCe1-A is required for the renal ammonia and K + response to hypokalemia

doi: 10.1152/ajprenal.00481.2019

Figure Lengend Snippet: Antibodies used (alphabetical order)

Article Snippet: ENaCβ , StressMarq Biosciences (SPC-404, Victoria, BC, Canada) , WB, 1:5,000.

Techniques: Generated

Journal: Acta Neuropathologica Communications

Article Title: Acute axon damage and demyelination are mitigated by 4-aminopyridine (4-AP) therapy after experimental traumatic brain injury

doi: 10.1186/s40478-022-01366-z

Figure Lengend Snippet: 4-AP treatment improves the molecular organization of excitable axonal domains that are disrupted by TBI. Confocal imaging analysis of node of Ranvier complexes in individual corpus callosum axons of Thy1-YFP-16 mice at 7 days post-TBI or sham procedures. ( A ) Immunostaining along YFP-labeled axons (green) detected clustering of voltage-gated sodium channels (Nav1.6, white) at the node and Caspr (red) in the paranode region, where myelin attaches to the axon. B-D TBI disrupted paranode domain organization, which was normalized by 4-AP treatment. ( B ) “Symmetrical” Caspr paranodes exhibit paired Caspr bands of approximately symmetrical length, while “Asymmetrical” paranodes exhibit uneven Caspr domain lengths. Single Caspr paranodes with a missing domain counterpart on the opposite side of a node were classified as “Heminodes”. C TBI increased the number of Caspr heminodes while acute 4-AP treatment post-TBI resulted in heminode numbers similar to sham levels. D TBI increased the asymmetry of Caspr domains in TBI mice compared to sham. 4-AP treatment significantly improved paranode organization following TBI. E–F Kv1.2 channel (red) and Caspr (white) immunostaining along individual YFP (green) axons in single confocal optical slices. E In injured mice, Kv1.2 channels mislocalize from the juxtaparanode domain into Caspr-labeled paranode domain. F Quantification of atypical Kv1.2 domains that overlapped with Caspr domains and/or were asymmetrical in length. Acute 4-AP treatment reduced the percentage of atypical Kv1.2 domains after TBI, but the distribution patterns of Kv1.2 channels was not fully normalized to sham levels. C, D, F Bars represent mean ± SEM with an individual data point shown for each mouse. Two-way ANOVA for main effect of injury or drug with Holm-Sidak’s multiple comparisons test for significance of post-hoc pair effects. See Fig. for mouse sample numbers and Table for statistical details

Article Snippet: Briefly, detection of Nav1.6 (Alomone Labs, Jerusalem, Israel; Cat# ASC-009, RRID:AB_2040202) at the nodes of Ranvier and Caspr (UC Davis/NIH NeuroMab Facility Cat# K65/35, RRID:AB_2877274) at adjacent paranodes.

Techniques: Imaging, Immunostaining, Labeling

Journal: Psychopharmacology

Article Title: Specificity of a rodent alpha(α)6 nicotinic acetylcholine receptor subunit antibody

doi: 10.1007/s00213-019-05413-x

Figure Lengend Snippet: Evaluation of an alpha(α)6 nicotinic acetylcholine receptor subunit antibody. A western blot of α6 nAChR subunit expression and antigen block in bilateral ventral tegmental tissue punches collected from male Sprague Dawley rats. n=2 animals total. GAPDH is used as a loading control.

Article Snippet: Although, a control antigen (Alomone) blocks the α6 nAChR subunit antibody from binding in the PC12 pheochromocytoma cells, a more standard form of validation is necessary ( Uhlen et al. 2016 ).

Techniques: Western Blot, Expressing, Blocking Assay

Journal: Psychopharmacology

Article Title: Specificity of a rodent alpha(α)6 nicotinic acetylcholine receptor subunit antibody

doi: 10.1007/s00213-019-05413-x

Figure Lengend Snippet: Genetic validation of an alpha(α)6 nicotinic acetylcholine receptor subunit antibody. A.) Western blot and B.) relative band intensity quantification of α6 nAChR subunit expression and antigen block from whole brain tissue collected from male and female WT and α6 KO C57BL/6J mice, n=4 animals/genotype; **p< 0.01 and ***p< 0.001 α6 antibody vs. α6 antibody + antigen normalized signal. GAPDH is used as a loading control.

Article Snippet: Although, a control antigen (Alomone) blocks the α6 nAChR subunit antibody from binding in the PC12 pheochromocytoma cells, a more standard form of validation is necessary ( Uhlen et al. 2016 ).

Techniques: Western Blot, Expressing, Blocking Assay

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Iron does not transcriptionally regulate ENaC alpha. The kidneys of 12-week-old WT or iron overloaded Hamp -/- mice were analyzed by real time PCR for gene expression of ENaC alpha. There was no significant difference in ENaC alpha gene expression of WT or spontaneously iron overloaded Hamp -/- mice under basal conditions ( A ). We confirmed that iron can regulate ENaC alpha gene transcription in mpkCCD cells, a cortical collecting duct cell line that is known to express ENaC alpha. 200 × 10 5 cells were serum-starved for 12 h and then cultured for 12 more hours in vehicle or FAC (100 uM). RNA was purified and then cDNA was synthesized. ENaC alpha gene expression was measured. Iron loading did not regulate ENaC alpha gene expression in mpkCCD cells ( B ), though it increased oxidative stress ( C ). One hundred uM FAC did not activate cell death program under the experimental conditions ( D ). Data from 2 independent experiments were pooled and analyzed. The statistical significance was determined by a 2-tailed Mann–Whitney test and is plotted as mean ± SEM. ** p < 0.001.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Purification, Synthesized, MANN-WHITNEY

Journal: Biology

Article Title: Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel

doi: 10.3390/biology11010123

Figure Lengend Snippet: Immunohistochemistry analysis of ENaC alpha and MARCKS protein expression in Hamp -/- (KO) mice or wild-type (WT) mice. ( A ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in WT control mice. ( B ) ENaC alpha (red) protein expression and MARCKS (green) protein expression in iron overloaded Hamp -/- mice. Data is representative of 3 mice in each group.

Article Snippet: The slides were then boiled in citrate buffer for 20 min (Vector Laboratories, Inc.; Burlingame, CA, USA) and washed for 3-min in type 1 water and then in 1X phosphate-buffered saline (1XPBS) (Corning; Manassas, VA, USA) for 5 min. After blocking with 2.5% normal horse serum (Vector Laboratories, Inc.) for 20 min the tissues were incubated for 60 min with a 1:500 dilution of primary rabbit polyclonal anti-MARCKS antibody (ab72459; Abcam, Boston, MA, USA) followed by a 60-min incubation with mouse anti-ENaC alpha antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-242D), mouse anti-ENaC beta antibody (StressMarq Biosciences Inc., Victoria BC, CA, Canada; SMC-240D) or anti-ENaC gamma 2102 antibody conjugated with the Dylight NHS ester 594 kit (ThermoFisher, Waltham, MA, USA).

Techniques: Immunohistochemistry, Expressing