sk3  (Alomone Labs)

Journal: bioRxiv

Article Title: Cross-species epigenetic regulation of nucleus accumbens KCNN3 transcript variants by excessive ethanol drinking and dependence

doi: 10.1101/713826

Figure Lengend Snippet: Summary of nucleus accumbens KCNN3 transcript expression in ethanol drinking rhesus macaques and C57BL/6J mice. a-c The relative expression of brain KCNN3 transcripts ( SK3_ex7/8 , 1 SK3_ex1B , and SK3_ex4 ) among the three drinking macaque groups ( SK3_ex1B : * p = 0.0381 vs CTRL; SK3_ex4 : ** p = 0.0084 vs CTRL, *** p = 0.0009 vs CTRL). d-f The relative expression of the KCNN3 transcripts in drinking monkeys collapsed by age at onset of ethanol drinking ( SK3_ex4 : ** p

Article Snippet: We first performed a series of western blots using different titrations of sample and antibody to establish the linear range for KCa 2.3 (Alomone Labs, Jerusalem, Israel; Catalog #: APC-025; epitope AA 2-21 of human KCa 2.3) in primate tissue samples.

Techniques: Expressing, Mouse Assay

Journal: bioRxiv

Article Title: Cross-species epigenetic regulation of nucleus accumbens KCNN3 transcript variants by excessive ethanol drinking and dependence

doi: 10.1101/713826

Figure Lengend Snippet: Adaptations in nucleus accumbens KCNN3 transcript and protein expression in ethanol drinking female rhesus macaques. a-c The relative expression of KCNN3 transcripts ( SK3_ex7/8 , 1 SK3_ex1B , and SK3_ex4 ) among control and very heavy drinking macaque groups ( SK3_ex1B : * p = 0.037 vs CTRL; SK3_ex4 : * p = 0.024 vs CTRL). d Characterization of anti-K Ca 2.3 channel western blot in macaque accumbens tissue (protein loading range, 1.25 – 40 µg). e Positive correlation between the amount of protein loaded and anti-K Ca 2.3 channel optical density values. f,g The full K Ca 2.3 channel blot and quantitation of normalized K Ca 2.3 channel protein expression in controls and drinkers (* p = 0.0324 vs CTRL).

Article Snippet: We first performed a series of western blots using different titrations of sample and antibody to establish the linear range for KCa 2.3 (Alomone Labs, Jerusalem, Israel; Catalog #: APC-025; epitope AA 2-21 of human KCa 2.3) in primate tissue samples.

Techniques: Expressing, Western Blot, Quantitation Assay

Journal: bioRxiv

Article Title: Cross-species epigenetic regulation of nucleus accumbens KCNN3 transcript variants by excessive ethanol drinking and dependence

doi: 10.1101/713826

Figure Lengend Snippet: KCNN3 methylation levels within MR-ex1-200 of ethanol drinking monkeys and dependent mice. The average methylation rates of individual CpGs included in the methylation region under study are shown. a Exon organization of the KCNN3 locus showing the location of exons and the dual CAG trinucleotide repeat arrays and methylated region in exon 1 (MR-ex1). b In rhesus macaque, the following CpGs showed elevated rates of methylation in heavy/very heavy drinking macaques vs controls: CpG 129130376 : F(2, 11.846) = 7.9, * p = 0.04; CpG 129130680 : F(2, 15.955) = 3.661, * p = 0.036; CpG 129130739 : F(2, 15.468) = 3.817, * p = 0.04; CpG 129130770 : F(2, 13.57) = 5.047, * p = 0.041; CpG 129130792 : F(2, 13.945) = 7.047, * p = 0.01; CpG 129130816 : F(2, 15.63) = 5.836, * p = 0.015; CpG 129130832 : F(2, 15.473) = 3.95, * p = 0.033; CpG 129130931 : F(2, 14.393) = 4.016, * p = 0.038; CpG 129130964 : F(2, 15.708) = 3.985, * p = 0.031. c In female macaques, elevated rates of methylation were observed at the following CpGs in very heavy drinking macaques vs controls: CpG 129130612 : F(1, 7) = 6.122, * p = 0.048; CpG 129130632 : F(1, 7) = 7.64, * p = 0.033; CpG 129130685 : F(1, 7) = 13.370, * p = 0.011; CpG 129130699 : F(1, 7) = 7.799, * p = 0.031. d In mouse accumbens, the following CpGs showed elevated rates of methylation between non-drinking mice and drinking dependent mice: Independent t-tests: CpG 89555945, t (16)= −2.946, *** p = 0.0009; CpG 89555974, t (17) = −2.860, * p = 0.011; CpG 89556220, t (17) = −2.206, * p = 0.042.

Article Snippet: We first performed a series of western blots using different titrations of sample and antibody to establish the linear range for KCa 2.3 (Alomone Labs, Jerusalem, Israel; Catalog #: APC-025; epitope AA 2-21 of human KCa 2.3) in primate tissue samples.

Techniques: Methylation, Mouse Assay

Journal: bioRxiv

Article Title: Cross-species epigenetic regulation of nucleus accumbens KCNN3 transcript variants by excessive ethanol drinking and dependence

doi: 10.1101/713826

Figure Lengend Snippet: KCNN3 -CAG n allele frequency distribution among male and female rhesus macaques. a The frequency distribution of (CAG) n alleles is shown for low drinkers (LD), binge drinkers (BD), heavy drinkers (HD), and very heavy drinkers (VHD). b Correlation between CAG repeat sum and average ethanol intake. c-e Correlations between CAG repeat sum and KCNN3 transcript expression in long-term drinking rhesus macaques.

Article Snippet: We first performed a series of western blots using different titrations of sample and antibody to establish the linear range for KCa 2.3 (Alomone Labs, Jerusalem, Israel; Catalog #: APC-025; epitope AA 2-21 of human KCa 2.3) in primate tissue samples.

Techniques: Expressing

Journal:

Article Title: Effects of methyl ?-cyclodextrin on EDHF responses in pig and rat arteries; association between SKCa channels and caveolin-rich domains

doi: 10.1038/sj.bjp.0707222

Figure Lengend Snippet: Anti-SK3, IK1 and caveolin-1 Western blot analysis of anti-caveolin-1 immunoprecipitates prepared from porcine coronary artery endothelium. Immunoprecipitation (IP) was performed with and without anti-caveolin-1 antibody (+ve and −ve,

Article Snippet: Antibodies used were anti-SK3 (APC-025; Alomone Laboratories, Jerusalem, Israel), anti-IK1 (a kind gift of Dr M Chen, GlaxoSmithKline), anti-caveolin 1 mAb (clone 2234; BD Transduction Laboratories, San Diego, CA, USA), anti-caveolin 1 pAb (sc-894, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-eNOS (610298, BD Transduction Laboratories).

Techniques: Western Blot, Immunoprecipitation

Journal: Journal of Neuroinflammation

Article Title: The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

doi: 10.1186/1742-2094-7-4

Figure Lengend Snippet: The SK blocker, apamin, does not affect microglial phagocytosis . Phagocytosis of fluorescein-labeled E. coli bacteria was quantified using a fluorescence plate reader (see Methods), expressed as mean±SEM of the number of cell cultures indicated on each bar. To generate a phagocytosis index, data were normalized to the mean fluorescence signal from untreated microglia that had phagocytosed E. coli . Phagocytosis was not affected by a high concentration of apamin (100 nM) that blocks cloned SK1, SK2 and SK3 channels. In control experiments, phagocytosis was prevented by the actin-polymerization inhibitor, 10 μM cytochalasin D (*** p

Article Snippet: SK3 immunoreactivity was detected with an anti-SK3 antibody (rabbit polyclonal, Alomone Labs, Jerusalem, Israel) that we previously validated by Western blots on microglia and rSK3-transfected CHO cells [ ].

Techniques: Labeling, Fluorescence, Concentration Assay, Clone Assay

Journal: Journal of Neuroinflammation

Article Title: The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

doi: 10.1186/1742-2094-7-4

Figure Lengend Snippet: SK3 immunoreactivity in the striatum 3 days after an ischemic stroke. A . The ischemic lesion (scale bar, 500 μm) shows increased staining with the microglia/macrophage marker, 'ionized calcium-binding adapter-1' (Iba-1; rabbit polyclonal, 1:1000; Cy3-conjugated donkey anti-rabbit secondary, 1:400). The high-magnification image from the core (inset; scale bar, 20 μm) shows examples of activated microglia/macrophages, co-labeled with the nuclear stain, DAPI. B, C . Sections from the edge and centre of the lesion were co-labeled with tomato lectin and anti-SK3 (as in Figure 6). Scale bars: 100 μm for the main images; 20 μm for the inset. In panel B, note the unlabeled white matter tracts at the edge of the lesion (asterisks) surrounded by areas of diffuse SK3 staining. In panel C, almost every activated microglia/macrophage (examples marked with arrows) is co-labeled with SK3 and tomato lectin. Note the intense surface SK3 labeling of activated microglia/macrophages (inset).

Article Snippet: SK3 immunoreactivity was detected with an anti-SK3 antibody (rabbit polyclonal, Alomone Labs, Jerusalem, Israel) that we previously validated by Western blots on microglia and rSK3-transfected CHO cells [ ].

Techniques: Staining, Marker, Binding Assay, Labeling

Journal: Journal of Neuroinflammation

Article Title: The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

doi: 10.1186/1742-2094-7-4

Figure Lengend Snippet: Changes in SK3 immunoreactivity and microglia/macrophages after an intracerebral hemorrhage (ICH) . Confocal images in A-C were labeled with OX-42 antibody (mouse monoclonal; 1:200) and a Cy3-conjugated secondary (donkey anti-mouse, 1:400). A . A ring of activated microglia/macrophages surrounds the hematoma at 7 days after an ICH: c, centre; e, edge; s, surrounding striatum (scale bar, 500 μm). B . In the normal striatum, there is widespread OX-42 labeling of 'resting' microglia (scale bar, 100 μm). The inset shows a typical ramified microglial cell with long processes and a small cell body (scale bar, 20 μm). C . At the edge of the hematoma 7 days after an ICH, microglia/macrophages transition to amoeboid cells with partially retracted processes further from the lesion (and inset at upper right) to round cells near the hematoma (and left inset). Scale bars: 100 μm for the main image and 20 μm for the insets. D . At the edge of the hematoma, SK3 labeling in microglia/macrophages (as in Figure 6) is shown in high-magnification color-separated images (scale bars, 10 μm). The negative control (inset in D' ) shows lack of staining with Cy3-conjugated 2° antibody alone. The merged image (D'') shows extensive surface SK3 staining in activated microglia/macrophages (examples marked with arrows).

Article Snippet: SK3 immunoreactivity was detected with an anti-SK3 antibody (rabbit polyclonal, Alomone Labs, Jerusalem, Israel) that we previously validated by Western blots on microglia and rSK3-transfected CHO cells [ ].

Techniques: Labeling, Negative Control, Staining

Journal: Journal of Neuroinflammation

Article Title: The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

doi: 10.1186/1742-2094-7-4

Figure Lengend Snippet: Blocking SK3 channels in microglia reduces their neurotoxic behavior . Microglia on Transwell™ inserts were incubated with lipopolysaccharide (LPS; 100 ng/ml, 24 h), with or without 100 nM apamin, 5 nM tamapin or 250 pM tamapin. The inserts were then washed to remove the drugs; thus, target neurons were never exposed to LPS or channel blockers. Each microglia-bearing insert was placed in a Transwell™ chamber above healthy neurons, incubated for 24 h (for caspase 3 activation) or 48 h (for TUNEL), and then the target neuron cultures were removed and assessed. Results are presented as mean±SEM for the number of separate cultures indicated on the bars. A . TUNEL-positive neuronal nuclei were counted and expressed as a percentage of all DAPI-stained nuclei. LPS-stimulated microglia killed more neurons than untreated microglia ( †† p

Article Snippet: SK3 immunoreactivity was detected with an anti-SK3 antibody (rabbit polyclonal, Alomone Labs, Jerusalem, Israel) that we previously validated by Western blots on microglia and rSK3-transfected CHO cells [ ].

Techniques: Blocking Assay, Incubation, Activation Assay, TUNEL Assay, Staining

Journal: Journal of Neuroinflammation

Article Title: The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

doi: 10.1186/1742-2094-7-4

Figure Lengend Snippet: Blocking SK3 channels reduces microglial iNOS and NO, and tyrosine nitration of target neurons . Microglia were grown on Transwell™ inserts, and treated with LPS (100 ng/ml, 24 h), with or without 100 nM apamin, 5 nM tamapin or 250 pM tamapin. Results are expressed as RFU/mg protein in each well (mean±SEM; # of cell cultures indicated on bars), normalized to the signal from untreated microglia. A . iNOS protein was monitored with a mouse monoclonal anti-iNOS antibody (1:200) and a Cy3-conjugated secondary antibody (1:500). LPS stimulation increased iNOS expression ( † p

Article Snippet: SK3 immunoreactivity was detected with an anti-SK3 antibody (rabbit polyclonal, Alomone Labs, Jerusalem, Israel) that we previously validated by Western blots on microglia and rSK3-transfected CHO cells [ ].

Techniques: Blocking Assay, Nitration, Expressing

Journal: Journal of Neuroinflammation

Article Title: The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

doi: 10.1186/1742-2094-7-4

Figure Lengend Snippet: Blocking microglial SK3 channels inhibits p38 MAPK (but not NFκB) activation . Microglia cultures were treated with LPS (100 ng/ml), with or without 100 nM apamin, 5 nM tamapin or 250 pM tamapin. p38 MAPK activation was monitored with a rabbit polyclonal antibody against phosphorylated (active) p38 MAPK (1:50). NF-κB activation was monitored as degradation of IκB-α, using a rabbit polyclonal antibody against IκB-α (1:100). Immunoreactivity was detected using a Cy3-conjugated secondary antibody (1:500). A . The phospho-p38 MAPK fluorescence signal was increased in microglia after 30 min lipopolysaccharide treatment († p

Article Snippet: SK3 immunoreactivity was detected with an anti-SK3 antibody (rabbit polyclonal, Alomone Labs, Jerusalem, Israel) that we previously validated by Western blots on microglia and rSK3-transfected CHO cells [ ].

Techniques: Blocking Assay, Activation Assay, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

doi: 10.1186/1742-2094-7-4

Figure Lengend Snippet: SK3 immunoreactivity in the healthy adult rat striatum . Confocal micrographs of coronal sections show salient features of the adult rat striatum, microglia/macrophages, and SK3 staining. A . A low-magnification image of the entire hemisphere (scale bar, 1 mm); the striatum is indicated by a dashed curve; V, ventricle; CC, corpus callosum. White matter is labeled with an antibody against myelin basic protein (MBP) (mouse monoclonal, 1:100) and a Cy3-conjugated secondary antibody (donkey anti-mouse, 1:400); note the dark 'holes' between the brightly stained axon bundles seen in cross-section. B . Diffuse SK3 staining is seen between the unstained white matter tracts (asterisks) in the normal striatum: co-labeled with FITC-conjugated tomato lectin (TL, 1:400), and anti-SK3 (rabbit polyclonal, 1:500) with a Cy3-conjugated secondary antibody (donkey anti-rabbit, 1:400); scale bar, 100 μm. Lack of non-specific staining is shown by the negative control (inset) with Cy3-conjugated 2° antibody alone. C . A high-magnification image (scale bar, 5 μm) shows intense SK3 staining in microglial processes (marked with arrows) and punctate staining elsewhere (labeled as above; and with the nuclear stain, DAPI).

Article Snippet: SK3 immunoreactivity was detected with an anti-SK3 antibody (rabbit polyclonal, Alomone Labs, Jerusalem, Israel) that we previously validated by Western blots on microglia and rSK3-transfected CHO cells [ ].

Techniques: Staining, Labeling, Negative Control

Journal: Journal of Neuroinflammation

Article Title: The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro

doi: 10.1186/1742-2094-7-4

Figure Lengend Snippet: KCNN /SK expression in cultured microglia . A . Relative mRNA expression of KCNN1 - KCNN3 was determined by quantitative real-time RT-PCR (qRT-PCR), and normalized to the housekeeping gene, TATA box-binding protein (TBP). Bars represent mean ± SEM for 3 cultures from separate rat litters. After microglia were activated with lipopolysaccharide (LPS; 100 ng/ml, 24 h), KCNN3 expression increased ( † p

Article Snippet: SK3 immunoreactivity was detected with an anti-SK3 antibody (rabbit polyclonal, Alomone Labs, Jerusalem, Israel) that we previously validated by Western blots on microglia and rSK3-transfected CHO cells [ ].

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Binding Assay

Journal: Pain

Article Title: In vivo activation of the SK channel in the spinal cord reduces the NMDA receptor antagonist dose needed to produce antinociception in an inflammatory pain model

doi: 10.1097/j.pain.0000000000000124

Figure Lengend Snippet: Postsynaptic expression of the SK channel subunit, SK3, is reduced after CFA. Average histograms and representative western blots showing protein expression levels of SK1 (lower band), SK2 and SK3 subunits in the total homogenate (TH) and postsynaptic

Article Snippet: After washing with PBS and blocking with 30% goat serum in PBS with 0.5% Triton X-100, sections were incubated overnight with SK3 (1:1000, Alomone-labs) antibody in PBS with 0.5% Triton X-100 at 4 °C or SK3 and NR1 (1:100, Invitrogen) antibodies in PBS Triton X-100 0.2% or SK3 and NeuN (1:5000, Millipore) antibodies in PBS Triton X-100 0.2%.

Techniques: Expressing, Western Blot

Journal: Pain

Article Title: In vivo activation of the SK channel in the spinal cord reduces the NMDA receptor antagonist dose needed to produce antinociception in an inflammatory pain model

doi: 10.1097/j.pain.0000000000000124

Figure Lengend Snippet: SK3 channels show both somatic and dendritic expression. A SK3 (green) immunolabeling shows abundant distribution throughout DH laminae (10x). Control (blank) image shows that there was no immunoreaction product in the spinal cord sections in the absence

Article Snippet: After washing with PBS and blocking with 30% goat serum in PBS with 0.5% Triton X-100, sections were incubated overnight with SK3 (1:1000, Alomone-labs) antibody in PBS with 0.5% Triton X-100 at 4 °C or SK3 and NR1 (1:100, Invitrogen) antibodies in PBS Triton X-100 0.2% or SK3 and NeuN (1:5000, Millipore) antibodies in PBS Triton X-100 0.2%.

Techniques: Expressing, Immunolabeling

Journal: Pain

Article Title: In vivo activation of the SK channel in the spinal cord reduces the NMDA receptor antagonist dose needed to produce antinociception in an inflammatory pain model

doi: 10.1097/j.pain.0000000000000124

Figure Lengend Snippet: SK3-containing SK channels can be co-expressed in close proximity with NMDA receptors. Double immunolabeling with SK3 (green) and NR1 (red) antibodies shows co-expression (yellow) of NR1 subunit of NMDAR and SK3-containing channels within the same neuron

Article Snippet: After washing with PBS and blocking with 30% goat serum in PBS with 0.5% Triton X-100, sections were incubated overnight with SK3 (1:1000, Alomone-labs) antibody in PBS with 0.5% Triton X-100 at 4 °C or SK3 and NR1 (1:100, Invitrogen) antibodies in PBS Triton X-100 0.2% or SK3 and NeuN (1:5000, Millipore) antibodies in PBS Triton X-100 0.2%.

Techniques: Immunolabeling, Expressing