sk3 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs sk3 antibody
    mRNA levels of <t>SK3</t> in the rat uterus of the different groups of rats. All data are ratio to β-actin in the same sample and sham values are set to 1. Data are presented as mean ± S.E.M. Asterisk (*) represent significant difference in the different groups of animals (p
    Sk3 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sk3 antibody/product/Alomone Labs
    Average 94 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Expression of the Small Conductance Ca2+-Activated Potassium Channel Subtype 3 (SK3) in Rat Uterus after Stimulation with 17?-Estradiol"

    Article Title: Expression of the Small Conductance Ca2+-Activated Potassium Channel Subtype 3 (SK3) in Rat Uterus after Stimulation with 17?-Estradiol

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087652

    mRNA levels of SK3 in the rat uterus of the different groups of rats. All data are ratio to β-actin in the same sample and sham values are set to 1. Data are presented as mean ± S.E.M. Asterisk (*) represent significant difference in the different groups of animals (p
    Figure Legend Snippet: mRNA levels of SK3 in the rat uterus of the different groups of rats. All data are ratio to β-actin in the same sample and sham values are set to 1. Data are presented as mean ± S.E.M. Asterisk (*) represent significant difference in the different groups of animals (p

    Techniques Used:

    SK3 protein expression in the four groups of animals. (A) Accumulated measurements of protein expression from all groups. All data are ratio to β-actin in the same sample. Data are presented as mean ± S.E.M. Asterisk (*) represent significant difference from the oveariectomized rats (*p
    Figure Legend Snippet: SK3 protein expression in the four groups of animals. (A) Accumulated measurements of protein expression from all groups. All data are ratio to β-actin in the same sample. Data are presented as mean ± S.E.M. Asterisk (*) represent significant difference from the oveariectomized rats (*p

    Techniques Used: Expressing

    Immuno staining of rat uterus in the four groups of animals showing localization of SK3. SK3 staining of sham-operated animals (A), of ovariectomized animals (B), of 17β-estradiol-treated animals (C) and of progesterone-treated animals (D). A–D is shown in 10x magnification. Inserts show 40x magnification of muscle layer (**) and epithelium (*). E–H) Absorptions control using specific blocking peptide. Positive SK3 staining in a 1∶8000 dilution showing signal in both epithelium and smooth muscle layer (E and G). Peptide absorptions control, a 1∶8000 dilution of antibody/control peptide complex eliminates the staining in epithelium and muscle (F and H). Controls are shown in 40x magnification. All scale bars represent 0.1 mm. Arrows point at epithelial localization, while arrow heads point at muscular localization.
    Figure Legend Snippet: Immuno staining of rat uterus in the four groups of animals showing localization of SK3. SK3 staining of sham-operated animals (A), of ovariectomized animals (B), of 17β-estradiol-treated animals (C) and of progesterone-treated animals (D). A–D is shown in 10x magnification. Inserts show 40x magnification of muscle layer (**) and epithelium (*). E–H) Absorptions control using specific blocking peptide. Positive SK3 staining in a 1∶8000 dilution showing signal in both epithelium and smooth muscle layer (E and G). Peptide absorptions control, a 1∶8000 dilution of antibody/control peptide complex eliminates the staining in epithelium and muscle (F and H). Controls are shown in 40x magnification. All scale bars represent 0.1 mm. Arrows point at epithelial localization, while arrow heads point at muscular localization.

    Techniques Used: Immunostaining, Staining, Blocking Assay

    2) Product Images from "SK3/TRPC1/Orai1 complex regulates SOCE-dependent colon cancer cell migration: a novel opportunity to modulate anti-EGFR mAb action by the alkyl-lipid Ohmline"

    Article Title: SK3/TRPC1/Orai1 complex regulates SOCE-dependent colon cancer cell migration: a novel opportunity to modulate anti-EGFR mAb action by the alkyl-lipid Ohmline

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8786

    Proposed mechanism demonstrating the interaction between the lipid-raft associated Orai1/TRPC1/SK3 channel complex and EGFR signaling pathway in colon cancer cell migration This model suggests three positive feedback loops: 1) STIM1 following its phosphorylation by EGF stimulation and Akt, is the trigger of SOCE and promotes migration mediated by the translocation of TRPC1 and Orai1 into lipid rafts where SK3 is concentrated. 2) The Orai1/TRPC1/SK3 channel complex promotes SOCE, which enhances P-Akt leading to the phosphorylation of STIM1, that may promote SOCE and 3) P-Akt activates Rac1, which enhances SOCE and in turn P-Akt. These loops operated toward the same goal: enhancing SOCE and SK3-dependent cell migration.
    Figure Legend Snippet: Proposed mechanism demonstrating the interaction between the lipid-raft associated Orai1/TRPC1/SK3 channel complex and EGFR signaling pathway in colon cancer cell migration This model suggests three positive feedback loops: 1) STIM1 following its phosphorylation by EGF stimulation and Akt, is the trigger of SOCE and promotes migration mediated by the translocation of TRPC1 and Orai1 into lipid rafts where SK3 is concentrated. 2) The Orai1/TRPC1/SK3 channel complex promotes SOCE, which enhances P-Akt leading to the phosphorylation of STIM1, that may promote SOCE and 3) P-Akt activates Rac1, which enhances SOCE and in turn P-Akt. These loops operated toward the same goal: enhancing SOCE and SK3-dependent cell migration.

    Techniques Used: Migration, Translocation Assay

    STIM1 activation, triggered by Ca2+ store depletion, recruits an Orai1/TRPC1 complex into lipid-rafts containing SK3 channels A. Left panel, Immunoblots representing membrane fractionation, on a sucrose gradient of cell lysate in control condition. SK3 is exclusively located into lipid-rafts whereas calcium channels Orai1 and TRPC1 are found outside lipid-rafts. Right panel, representative confocal images of SK3 and Caveolin-1 staining in HCT-116 cells showing immunocolocalization of SK3 and Caveolin-1. B. Depletion of intracellular calcium store by Tg induces the translocation of calcium channels Orai1 and TRPC1 into lipid-rafts whereas SK3 is always in lipid-rafts. Immunoblots representing membrane fractionation, on a sucrose gradient, of cells treated with 5μM Tg for 20 min. C. TRPC1, Orai1, STIM1 and caveolin-1 form a lipid-raft complex in HCT-116 after Ca 2+ store depletion. Immunoblots depict co-immunoprecipitation between STIM1, TRPC1, Orai1 and Caveolin-1 before and after Ca 2+ store depletion.
    Figure Legend Snippet: STIM1 activation, triggered by Ca2+ store depletion, recruits an Orai1/TRPC1 complex into lipid-rafts containing SK3 channels A. Left panel, Immunoblots representing membrane fractionation, on a sucrose gradient of cell lysate in control condition. SK3 is exclusively located into lipid-rafts whereas calcium channels Orai1 and TRPC1 are found outside lipid-rafts. Right panel, representative confocal images of SK3 and Caveolin-1 staining in HCT-116 cells showing immunocolocalization of SK3 and Caveolin-1. B. Depletion of intracellular calcium store by Tg induces the translocation of calcium channels Orai1 and TRPC1 into lipid-rafts whereas SK3 is always in lipid-rafts. Immunoblots representing membrane fractionation, on a sucrose gradient, of cells treated with 5μM Tg for 20 min. C. TRPC1, Orai1, STIM1 and caveolin-1 form a lipid-raft complex in HCT-116 after Ca 2+ store depletion. Immunoblots depict co-immunoprecipitation between STIM1, TRPC1, Orai1 and Caveolin-1 before and after Ca 2+ store depletion.

    Techniques Used: Activation Assay, Western Blot, Fractionation, Staining, Translocation Assay, Immunoprecipitation

    Ohmline as a new personalized treatment strategy to decrease P-Akt and therefore modulate the effects of Anti-EGFR mAbs A. Disrupting lipid-rafts with the alkyl-lipid Ohmline allows SK3 to re-translocate outside away from lipid-rafts without modifying the localization of calcium channels whereas the co-treatment Ohmline/Tg the translocation of calcium channels into lipid-raft. Immunoblots representing membrane fractionation, on a sucrose gradient, of cells treated with Ohmline alone (middle panel) or associated with 5μM Tg for 20 min (right panel). B. Left panel, Dissociation of the lipid-raft Orai1/TRPC1/SK3 channel complex by Ohmline decreased Ca 2+ entry evoked by Tg. Fluorescence measurement and relative fluorescence of Ca 2+ entry after intracellular calcium store depletion by Tg in cells treated 24h with Ohmline. Data represent means ± SEM. *p
    Figure Legend Snippet: Ohmline as a new personalized treatment strategy to decrease P-Akt and therefore modulate the effects of Anti-EGFR mAbs A. Disrupting lipid-rafts with the alkyl-lipid Ohmline allows SK3 to re-translocate outside away from lipid-rafts without modifying the localization of calcium channels whereas the co-treatment Ohmline/Tg the translocation of calcium channels into lipid-raft. Immunoblots representing membrane fractionation, on a sucrose gradient, of cells treated with Ohmline alone (middle panel) or associated with 5μM Tg for 20 min (right panel). B. Left panel, Dissociation of the lipid-raft Orai1/TRPC1/SK3 channel complex by Ohmline decreased Ca 2+ entry evoked by Tg. Fluorescence measurement and relative fluorescence of Ca 2+ entry after intracellular calcium store depletion by Tg in cells treated 24h with Ohmline. Data represent means ± SEM. *p

    Techniques Used: Translocation Assay, Western Blot, Fractionation, Fluorescence

    Migration of colon cancer cells HCT-116 is dependent on calcium-activated potassium channel SK3 and SOCE A. SK3 channel is involved in HCT-116 cell migration. Histograms showing HCT-116 cell migration transfected for 48h with siSK3 or treated with Apamin. The normalized cell number corresponds to the ratio of total number of migrating cells in presence of drugs/total number of migrating cells in control experiments. Results are expressed as mean ± SEM. **p
    Figure Legend Snippet: Migration of colon cancer cells HCT-116 is dependent on calcium-activated potassium channel SK3 and SOCE A. SK3 channel is involved in HCT-116 cell migration. Histograms showing HCT-116 cell migration transfected for 48h with siSK3 or treated with Apamin. The normalized cell number corresponds to the ratio of total number of migrating cells in presence of drugs/total number of migrating cells in control experiments. Results are expressed as mean ± SEM. **p

    Techniques Used: Migration, Transfection

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    Alomone Labs anti sk3
    Localization of <t>SK3</t> and SK2 protein in porcine coronary arteries by immunofluorescence labelling and Western blotting. (A), positive identification by von Willebrand's factor immunoreactivity (red) of the endothelium (Endo.) in a cross-section. Positions of the lumen, internal elastic lamina (IEL, green), smooth muscle (S. Muscle) and nuclei (blue) are shown. (B), cross-section labelled for SK3 (red) and nuclei (blue), with IEL (green) visible (inset, primary antibody pre-incubated with control peptide). (C), arteries opened longitudinally allowing the endothelium to be viewed en-face were labelled for SK3 (green) and nuclei (red) (inset, primary antibody omitted). (D), cross-section labelled for SK2 (red) and nuclei (blue), with IEL (green) shown (inset, primary antibody pre-incubated with control peptide). (E), endothelium viewed en-face and labelled for SK2 (green) and nuclei (red) (inset, primary antibody omitted). Areas where green SK2 label and red nuclei overlap appear yellow or orange. All scale bars are 25 μm. (F), Western blot analysis of SK3 and SK2 protein expression. Whole artery samples were separated into post-nuclear (P) and nuclear (N) fractions. Additional samples were prepared from endothelium (Endo.) only. Primary antibodies were used with (+) and without (−) control peptide pre-incubation. Molecular weight markers are indicated (kDa). 10 μg protein loadings.
    Anti Sk3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Alomone Labs rabbit anti sk3
    Expression of <t>SK3</t> channels, Abi-1 and nWASP in neuronal stem cells (NSCs) and in primary hippocampal neurons (HNs). (A) Western Blot analysis of SK3 channel transfected NSCs (SK3 pIRES) indicates that the SK3 channel antibody readily recognizes the overexpressed protein. NSCs transfected with the RNAi construct show a decrease of SK3 protein compared to NSCs transfected with a scrambled RNAi construct and untransfected cells. The SK3 channel protein can also be detected in hippocampal neurons. Subfractionation of brain tissue reveals that the protein is enriched towards the postsynaptic density fraction (PSD). (B) nWASP and Abi-1 are detectable in NSCs as well as in hippocampal neurons. (C) Light cycler analysis of SK3 channel mRNA in NSCs and hippocampal neurons reveals that SK3 transcript levels are downregulated after induction of differentiation in NSCs but show a steep increase in later steps of hippocampal neuron maturation. P -values from ANOVA for multiple-group comparison are 0.0011 for NSCs and
    Rabbit Anti Sk3, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti kca2 3 atto 594
    Expression of CD140α, CD44, CD34 and <t>SK3</t> cell surface markers in iMSCs, fibroblasts, and PDGFRα-positive cells. Flow cytometry analysis detected the expression of CD140α (( A ). cell count; ( B) . mean fluorescence intensity), CD44 (( C ). cell count; ( D ). mean fluorescence intensity), CD34 (( E ). cell count; ( F ). mean fluorescence intensity), and SK3 (( G ). cell count; ( H ). mean fluorescence intensity) in iMSCs, fibroblasts, and PDGFRα-positive ( n = 3, * p
    Anti Kca2 3 Atto 594, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Localization of SK3 and SK2 protein in porcine coronary arteries by immunofluorescence labelling and Western blotting. (A), positive identification by von Willebrand's factor immunoreactivity (red) of the endothelium (Endo.) in a cross-section. Positions of the lumen, internal elastic lamina (IEL, green), smooth muscle (S. Muscle) and nuclei (blue) are shown. (B), cross-section labelled for SK3 (red) and nuclei (blue), with IEL (green) visible (inset, primary antibody pre-incubated with control peptide). (C), arteries opened longitudinally allowing the endothelium to be viewed en-face were labelled for SK3 (green) and nuclei (red) (inset, primary antibody omitted). (D), cross-section labelled for SK2 (red) and nuclei (blue), with IEL (green) shown (inset, primary antibody pre-incubated with control peptide). (E), endothelium viewed en-face and labelled for SK2 (green) and nuclei (red) (inset, primary antibody omitted). Areas where green SK2 label and red nuclei overlap appear yellow or orange. All scale bars are 25 μm. (F), Western blot analysis of SK3 and SK2 protein expression. Whole artery samples were separated into post-nuclear (P) and nuclear (N) fractions. Additional samples were prepared from endothelium (Endo.) only. Primary antibodies were used with (+) and without (−) control peptide pre-incubation. Molecular weight markers are indicated (kDa). 10 μg protein loadings.

    Journal: British Journal of Pharmacology

    Article Title: Characterization of an apamin-sensitive small-conductance Ca2+-activated K+ channel in porcine coronary artery endothelium: relevance to EDHF

    doi: 10.1038/sj.bjp.0704551

    Figure Lengend Snippet: Localization of SK3 and SK2 protein in porcine coronary arteries by immunofluorescence labelling and Western blotting. (A), positive identification by von Willebrand's factor immunoreactivity (red) of the endothelium (Endo.) in a cross-section. Positions of the lumen, internal elastic lamina (IEL, green), smooth muscle (S. Muscle) and nuclei (blue) are shown. (B), cross-section labelled for SK3 (red) and nuclei (blue), with IEL (green) visible (inset, primary antibody pre-incubated with control peptide). (C), arteries opened longitudinally allowing the endothelium to be viewed en-face were labelled for SK3 (green) and nuclei (red) (inset, primary antibody omitted). (D), cross-section labelled for SK2 (red) and nuclei (blue), with IEL (green) shown (inset, primary antibody pre-incubated with control peptide). (E), endothelium viewed en-face and labelled for SK2 (green) and nuclei (red) (inset, primary antibody omitted). Areas where green SK2 label and red nuclei overlap appear yellow or orange. All scale bars are 25 μm. (F), Western blot analysis of SK3 and SK2 protein expression. Whole artery samples were separated into post-nuclear (P) and nuclear (N) fractions. Additional samples were prepared from endothelium (Endo.) only. Primary antibodies were used with (+) and without (−) control peptide pre-incubation. Molecular weight markers are indicated (kDa). 10 μg protein loadings.

    Article Snippet: Anti-SK2 and anti-SK3 (Alomone Labs), supplied with control peptides, anti-von Willebrand's factor (Novocastra) and secondary antibody conjugates (Jackson Immunoresearch) were used.

    Techniques: Immunofluorescence, Western Blot, Incubation, Expressing, Molecular Weight

    Detection of SK Ca subunit mRNA in porcine coronary artery endothelium. (A), RT – PCR analysis of GAPDH (G), SK1, SK2 and SK3 expression in samples of endothelium (Endo.) and positive control pig brain prepared with (+) and without (−) reverse transcription. Size markers in base pairs are indicated. In endothelium cDNA, GAPDH ( n =5/5), SK3 ( n =5/5) and SK2 ( n =3/5) were detected but not SK1 ( n =5). (B), the complete SK3 sequence from porcine coronary artery endothelium was determined by RT – PCR and 5′ and 3′ RACE. Start and stop codons are underlined.

    Journal: British Journal of Pharmacology

    Article Title: Characterization of an apamin-sensitive small-conductance Ca2+-activated K+ channel in porcine coronary artery endothelium: relevance to EDHF

    doi: 10.1038/sj.bjp.0704551

    Figure Lengend Snippet: Detection of SK Ca subunit mRNA in porcine coronary artery endothelium. (A), RT – PCR analysis of GAPDH (G), SK1, SK2 and SK3 expression in samples of endothelium (Endo.) and positive control pig brain prepared with (+) and without (−) reverse transcription. Size markers in base pairs are indicated. In endothelium cDNA, GAPDH ( n =5/5), SK3 ( n =5/5) and SK2 ( n =3/5) were detected but not SK1 ( n =5). (B), the complete SK3 sequence from porcine coronary artery endothelium was determined by RT – PCR and 5′ and 3′ RACE. Start and stop codons are underlined.

    Article Snippet: Anti-SK2 and anti-SK3 (Alomone Labs), supplied with control peptides, anti-von Willebrand's factor (Novocastra) and secondary antibody conjugates (Jackson Immunoresearch) were used.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Sequencing

    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

    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

    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).

    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

    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.

    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

    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.

    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

    Expression of SK3 channels, Abi-1 and nWASP in neuronal stem cells (NSCs) and in primary hippocampal neurons (HNs). (A) Western Blot analysis of SK3 channel transfected NSCs (SK3 pIRES) indicates that the SK3 channel antibody readily recognizes the overexpressed protein. NSCs transfected with the RNAi construct show a decrease of SK3 protein compared to NSCs transfected with a scrambled RNAi construct and untransfected cells. The SK3 channel protein can also be detected in hippocampal neurons. Subfractionation of brain tissue reveals that the protein is enriched towards the postsynaptic density fraction (PSD). (B) nWASP and Abi-1 are detectable in NSCs as well as in hippocampal neurons. (C) Light cycler analysis of SK3 channel mRNA in NSCs and hippocampal neurons reveals that SK3 transcript levels are downregulated after induction of differentiation in NSCs but show a steep increase in later steps of hippocampal neuron maturation. P -values from ANOVA for multiple-group comparison are 0.0011 for NSCs and

    Journal: PLoS ONE

    Article Title: An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

    doi: 10.1371/journal.pone.0018148

    Figure Lengend Snippet: Expression of SK3 channels, Abi-1 and nWASP in neuronal stem cells (NSCs) and in primary hippocampal neurons (HNs). (A) Western Blot analysis of SK3 channel transfected NSCs (SK3 pIRES) indicates that the SK3 channel antibody readily recognizes the overexpressed protein. NSCs transfected with the RNAi construct show a decrease of SK3 protein compared to NSCs transfected with a scrambled RNAi construct and untransfected cells. The SK3 channel protein can also be detected in hippocampal neurons. Subfractionation of brain tissue reveals that the protein is enriched towards the postsynaptic density fraction (PSD). (B) nWASP and Abi-1 are detectable in NSCs as well as in hippocampal neurons. (C) Light cycler analysis of SK3 channel mRNA in NSCs and hippocampal neurons reveals that SK3 transcript levels are downregulated after induction of differentiation in NSCs but show a steep increase in later steps of hippocampal neuron maturation. P -values from ANOVA for multiple-group comparison are 0.0011 for NSCs and

    Article Snippet: The following primary antibodies were used: rabbit anti-SK3 diluted 1∶200, rabbit (Alomone Labs), mouse anti-Nestin monoclonal diluted 1∶500 (BD Biosciences), mouse anti-Abi-1 diluted 1∶250 (MBL), mouse anti-myc antibody diluted 1∶500 (Invitrogen), rabbit anti-PSD95 diluted 1∶1000, rabbit anti-SV2 diluted 1∶300 (both abcam, Cambridge, USA) and rabbit anti-nWASP diluted 1∶500 (Santa Cruz) or chicken anti-nWASP diluted 1∶500 (abcam); fluorescence labeled secondary antibodies were Alexa Fluor® 488 (green, used filter set: excitation BP 450 – 490, FT 510, emission BP 515 - 565), Alexa Fluor® 568 (red, used filter set: excitation BP 534 nm–558 nm, FT 560, emission BP 575 - 640) and Alexa Fluor® 647 (magenta used filter set: excitation BP 610 nm–670 nm, FT 660, emission BP 640–740 (all from Invitrogen) all diluted 1∶500.

    Techniques: Expressing, Western Blot, Transfection, Construct

    Pharmacological treatment of NSCs with 1-EBIO, wiskostatin and apamin. Application of the SK3 channel opening substance 1-EBIO shows the rapid outgrowth and enlargement of filopodia with a translocation of the channel into the newly build processes in SK3 channel overexpressing NSCs. Apamin treatment that leads to a blockage of SK3 channels as well as the application of wiskostatin drastically reduces filopodia formation. (B) Double transfection of NSCs with SK3 channels and Abi-1 results in the co-localization of both proteins in the cell cytoplasm. The application of wiskostatin and apamin to the cells induced the translocation of the proteins to small microcompartments. In contrast, the SK3 channel activator 1-EBIO induced the rapid outgrowth of numerous large lamellipodia that show a netlike arrangement of SK3 channel immunoreactivity. (C) The double transfection of SK3 channels and nWASP resulted in a morphology of NSCs that was characterized by larger filopodial extensions that were lost after application of wiskostatin, enhanced and more elaborated under the influence of 1-EBIO and altered towards lamellipodia after apamin application. Scale bars as indicated.

    Journal: PLoS ONE

    Article Title: An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

    doi: 10.1371/journal.pone.0018148

    Figure Lengend Snippet: Pharmacological treatment of NSCs with 1-EBIO, wiskostatin and apamin. Application of the SK3 channel opening substance 1-EBIO shows the rapid outgrowth and enlargement of filopodia with a translocation of the channel into the newly build processes in SK3 channel overexpressing NSCs. Apamin treatment that leads to a blockage of SK3 channels as well as the application of wiskostatin drastically reduces filopodia formation. (B) Double transfection of NSCs with SK3 channels and Abi-1 results in the co-localization of both proteins in the cell cytoplasm. The application of wiskostatin and apamin to the cells induced the translocation of the proteins to small microcompartments. In contrast, the SK3 channel activator 1-EBIO induced the rapid outgrowth of numerous large lamellipodia that show a netlike arrangement of SK3 channel immunoreactivity. (C) The double transfection of SK3 channels and nWASP resulted in a morphology of NSCs that was characterized by larger filopodial extensions that were lost after application of wiskostatin, enhanced and more elaborated under the influence of 1-EBIO and altered towards lamellipodia after apamin application. Scale bars as indicated.

    Article Snippet: The following primary antibodies were used: rabbit anti-SK3 diluted 1∶200, rabbit (Alomone Labs), mouse anti-Nestin monoclonal diluted 1∶500 (BD Biosciences), mouse anti-Abi-1 diluted 1∶250 (MBL), mouse anti-myc antibody diluted 1∶500 (Invitrogen), rabbit anti-PSD95 diluted 1∶1000, rabbit anti-SV2 diluted 1∶300 (both abcam, Cambridge, USA) and rabbit anti-nWASP diluted 1∶500 (Santa Cruz) or chicken anti-nWASP diluted 1∶500 (abcam); fluorescence labeled secondary antibodies were Alexa Fluor® 488 (green, used filter set: excitation BP 450 – 490, FT 510, emission BP 515 - 565), Alexa Fluor® 568 (red, used filter set: excitation BP 534 nm–558 nm, FT 560, emission BP 575 - 640) and Alexa Fluor® 647 (magenta used filter set: excitation BP 610 nm–670 nm, FT 660, emission BP 640–740 (all from Invitrogen) all diluted 1∶500.

    Techniques: Translocation Assay, Transfection

    Structure and expression of the SK3 channel. (A,B) Domain structure and amino acid sequence of the rat SK3 channel. An N-terminal proline rich region (green) is indicated that harbors the interaction domain with the SH3 domain of Abi-1 (red). Bold letters indicate the transmembrane domains that are organized around the pore region (yellow). At the C-terminus the calmodulin binding site (light blue) as well as a coiled coil domain (purple) with a leucine zipper motif (purple in black frame) is located. (C) In situ hybridization experiments of a whole body embryo section at developmental stage day 20 and during rat brain development show that the SK3 channel transcripts are nearly exclusively expressed in brain. In embryonic tissue the mRNA is especially found in the subventricular zone, during brain development the transcripts are densely expressed in the dentate gyrus, the olfactory bulb, caudate putamen and in thalamic nuclei. (D) This schematic drawing illustrates the intramembranous localization of the protein showing that both the C- and the N-terminus are intracellular.

    Journal: PLoS ONE

    Article Title: An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

    doi: 10.1371/journal.pone.0018148

    Figure Lengend Snippet: Structure and expression of the SK3 channel. (A,B) Domain structure and amino acid sequence of the rat SK3 channel. An N-terminal proline rich region (green) is indicated that harbors the interaction domain with the SH3 domain of Abi-1 (red). Bold letters indicate the transmembrane domains that are organized around the pore region (yellow). At the C-terminus the calmodulin binding site (light blue) as well as a coiled coil domain (purple) with a leucine zipper motif (purple in black frame) is located. (C) In situ hybridization experiments of a whole body embryo section at developmental stage day 20 and during rat brain development show that the SK3 channel transcripts are nearly exclusively expressed in brain. In embryonic tissue the mRNA is especially found in the subventricular zone, during brain development the transcripts are densely expressed in the dentate gyrus, the olfactory bulb, caudate putamen and in thalamic nuclei. (D) This schematic drawing illustrates the intramembranous localization of the protein showing that both the C- and the N-terminus are intracellular.

    Article Snippet: The following primary antibodies were used: rabbit anti-SK3 diluted 1∶200, rabbit (Alomone Labs), mouse anti-Nestin monoclonal diluted 1∶500 (BD Biosciences), mouse anti-Abi-1 diluted 1∶250 (MBL), mouse anti-myc antibody diluted 1∶500 (Invitrogen), rabbit anti-PSD95 diluted 1∶1000, rabbit anti-SV2 diluted 1∶300 (both abcam, Cambridge, USA) and rabbit anti-nWASP diluted 1∶500 (Santa Cruz) or chicken anti-nWASP diluted 1∶500 (abcam); fluorescence labeled secondary antibodies were Alexa Fluor® 488 (green, used filter set: excitation BP 450 – 490, FT 510, emission BP 515 - 565), Alexa Fluor® 568 (red, used filter set: excitation BP 534 nm–558 nm, FT 560, emission BP 575 - 640) and Alexa Fluor® 647 (magenta used filter set: excitation BP 610 nm–670 nm, FT 660, emission BP 640–740 (all from Invitrogen) all diluted 1∶500.

    Techniques: Expressing, Sequencing, Binding Assay, In Situ Hybridization

    SK3 channel, nWASP and Abi-1 protein levels also alter hippocampal neuron morphology. (A) In close homology to the observation in NSCs we found that the overexpression of SK3 channels as well as of nWASP in developing hippocampal neurons is leading to a very complex dendritic tree with numerous secondary and tertiary dendrites. In contrast the Abi-1-GFP fusion protein extremely simplifies the dendritic arbor. (B-C) Overexpression of SK3 channels and Abi-1, nWASP respectively, resulted in a perfect co-localization of both fusion proteins especially in outgrowing neurites. Co-expression of SK3 channels and Abi-1 abolishes the SK3 channel overexpression phenotype while the addition of nWASP to SK3 channel transfected neurons is leading to a multidendritic neuron that is characterized by numerous primary, secondary and tertiary neurites. Nuclei are stained with DAPI (blue). Scale bars as indicated.

    Journal: PLoS ONE

    Article Title: An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

    doi: 10.1371/journal.pone.0018148

    Figure Lengend Snippet: SK3 channel, nWASP and Abi-1 protein levels also alter hippocampal neuron morphology. (A) In close homology to the observation in NSCs we found that the overexpression of SK3 channels as well as of nWASP in developing hippocampal neurons is leading to a very complex dendritic tree with numerous secondary and tertiary dendrites. In contrast the Abi-1-GFP fusion protein extremely simplifies the dendritic arbor. (B-C) Overexpression of SK3 channels and Abi-1, nWASP respectively, resulted in a perfect co-localization of both fusion proteins especially in outgrowing neurites. Co-expression of SK3 channels and Abi-1 abolishes the SK3 channel overexpression phenotype while the addition of nWASP to SK3 channel transfected neurons is leading to a multidendritic neuron that is characterized by numerous primary, secondary and tertiary neurites. Nuclei are stained with DAPI (blue). Scale bars as indicated.

    Article Snippet: The following primary antibodies were used: rabbit anti-SK3 diluted 1∶200, rabbit (Alomone Labs), mouse anti-Nestin monoclonal diluted 1∶500 (BD Biosciences), mouse anti-Abi-1 diluted 1∶250 (MBL), mouse anti-myc antibody diluted 1∶500 (Invitrogen), rabbit anti-PSD95 diluted 1∶1000, rabbit anti-SV2 diluted 1∶300 (both abcam, Cambridge, USA) and rabbit anti-nWASP diluted 1∶500 (Santa Cruz) or chicken anti-nWASP diluted 1∶500 (abcam); fluorescence labeled secondary antibodies were Alexa Fluor® 488 (green, used filter set: excitation BP 450 – 490, FT 510, emission BP 515 - 565), Alexa Fluor® 568 (red, used filter set: excitation BP 534 nm–558 nm, FT 560, emission BP 575 - 640) and Alexa Fluor® 647 (magenta used filter set: excitation BP 610 nm–670 nm, FT 660, emission BP 640–740 (all from Invitrogen) all diluted 1∶500.

    Techniques: Over Expression, Expressing, Transfection, Staining

    SK3 channels, Abi-1 and nWASP are found in neuronal complexes and colocalize in defined subcompartments of NSCs and hippocampal neurons (HNs). (A-B)Triple staining experiments show that Abi-1 as well as nWASP (red) colocalize with the SK3 channel (green) in distinct lamellipodia like structures of NSCs that are also rich of actin fibers stained by phalloidin (magenta), DAPI (blue) is used to label nuclei. (C–D) In hippocampal neurons SK3 channels are densely targeted to dendritic spines/PSDs and co-localize with nWASP as well as with Abi-1 (see arrows in insets). To visualize the cytoskeleton tubulin antibodies (Tubb, magenta) are used, DAPI staining (blue) show neuronal nuclei. (E) Co-immunoprecipitation experiments show the presence of all three molecules in one neuronal complex also in vivo . Magnetic beads loaded with SK3 channels antibodies were employed to precipitate the SK3 channel complex from rat brain. Within this precipitate Abi-1 as well as nWASP can be detected. (F) Schematic drawing of the fusions proteins and Abi-1 deletion constructs used for transfection experiments with their respective domain regions. The SK3 channel codes for a conserved N-terminal proline rich stretch that mediates the interaction with the Abi-1 SH3 domain. (G) Co-transfection of the nWASP with the SK3 channel results in a complete colocalization in NSCs. This is also observed when full length Abi-1-myc is co-transfected with the SK3 channel GFP fusion protein. The co-expression of SK3 channels with an Abi-1 SH3 domain deletion construct (Abi-1 ΔSH3-RFP) results in no co-localization of the proteins while the SK3 channel perfectly co-localizes with the Abi-1-SH3 domain fusion protein. Moreover, the full length SK3 channel protein can co-precipitate the GFP-SH3 domain from the co-transfected cell lysate but fails to bind to the Abi-1 protein that is not expressing the SH3 domain. Scale bars as indicated.

    Journal: PLoS ONE

    Article Title: An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

    doi: 10.1371/journal.pone.0018148

    Figure Lengend Snippet: SK3 channels, Abi-1 and nWASP are found in neuronal complexes and colocalize in defined subcompartments of NSCs and hippocampal neurons (HNs). (A-B)Triple staining experiments show that Abi-1 as well as nWASP (red) colocalize with the SK3 channel (green) in distinct lamellipodia like structures of NSCs that are also rich of actin fibers stained by phalloidin (magenta), DAPI (blue) is used to label nuclei. (C–D) In hippocampal neurons SK3 channels are densely targeted to dendritic spines/PSDs and co-localize with nWASP as well as with Abi-1 (see arrows in insets). To visualize the cytoskeleton tubulin antibodies (Tubb, magenta) are used, DAPI staining (blue) show neuronal nuclei. (E) Co-immunoprecipitation experiments show the presence of all three molecules in one neuronal complex also in vivo . Magnetic beads loaded with SK3 channels antibodies were employed to precipitate the SK3 channel complex from rat brain. Within this precipitate Abi-1 as well as nWASP can be detected. (F) Schematic drawing of the fusions proteins and Abi-1 deletion constructs used for transfection experiments with their respective domain regions. The SK3 channel codes for a conserved N-terminal proline rich stretch that mediates the interaction with the Abi-1 SH3 domain. (G) Co-transfection of the nWASP with the SK3 channel results in a complete colocalization in NSCs. This is also observed when full length Abi-1-myc is co-transfected with the SK3 channel GFP fusion protein. The co-expression of SK3 channels with an Abi-1 SH3 domain deletion construct (Abi-1 ΔSH3-RFP) results in no co-localization of the proteins while the SK3 channel perfectly co-localizes with the Abi-1-SH3 domain fusion protein. Moreover, the full length SK3 channel protein can co-precipitate the GFP-SH3 domain from the co-transfected cell lysate but fails to bind to the Abi-1 protein that is not expressing the SH3 domain. Scale bars as indicated.

    Article Snippet: The following primary antibodies were used: rabbit anti-SK3 diluted 1∶200, rabbit (Alomone Labs), mouse anti-Nestin monoclonal diluted 1∶500 (BD Biosciences), mouse anti-Abi-1 diluted 1∶250 (MBL), mouse anti-myc antibody diluted 1∶500 (Invitrogen), rabbit anti-PSD95 diluted 1∶1000, rabbit anti-SV2 diluted 1∶300 (both abcam, Cambridge, USA) and rabbit anti-nWASP diluted 1∶500 (Santa Cruz) or chicken anti-nWASP diluted 1∶500 (abcam); fluorescence labeled secondary antibodies were Alexa Fluor® 488 (green, used filter set: excitation BP 450 – 490, FT 510, emission BP 515 - 565), Alexa Fluor® 568 (red, used filter set: excitation BP 534 nm–558 nm, FT 560, emission BP 575 - 640) and Alexa Fluor® 647 (magenta used filter set: excitation BP 610 nm–670 nm, FT 660, emission BP 640–740 (all from Invitrogen) all diluted 1∶500.

    Techniques: Staining, Immunoprecipitation, In Vivo, Magnetic Beads, Construct, Transfection, Cotransfection, Expressing

    SK3 channel, nWASP and Abi-1 protein levels alter NSC morphology. (A–G) Overexpression of SK3 channels in NSCs significantly triggers long filopodia formation in NSCs while Abi-1 overexpression downregulates filopodia number and length (B). (C) Co-expression of SK3 channels and Abi-1 results in an NSC morphology that is identical to Abi-1 overexpression alone. (D) nWASP transfection strongly induces filopodia formation and this effect can be even enhanced by double transfection of nWASP together with SK3 channels (E). Downregulation of SK3 channels in NSCs by an RNAi construct that efficiently diminishes SK3 channel protein concentrations (F) does not significantly influence filopodia number and/or length of filopodia (G). Scale bars as indicated. (H–I) Statistical analysis of NSC morphology (number of filopodia per 1 µm/length of filopodia) after the above described transfection experiments. P -values from ANOVA for multiple-group comparison are

    Journal: PLoS ONE

    Article Title: An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

    doi: 10.1371/journal.pone.0018148

    Figure Lengend Snippet: SK3 channel, nWASP and Abi-1 protein levels alter NSC morphology. (A–G) Overexpression of SK3 channels in NSCs significantly triggers long filopodia formation in NSCs while Abi-1 overexpression downregulates filopodia number and length (B). (C) Co-expression of SK3 channels and Abi-1 results in an NSC morphology that is identical to Abi-1 overexpression alone. (D) nWASP transfection strongly induces filopodia formation and this effect can be even enhanced by double transfection of nWASP together with SK3 channels (E). Downregulation of SK3 channels in NSCs by an RNAi construct that efficiently diminishes SK3 channel protein concentrations (F) does not significantly influence filopodia number and/or length of filopodia (G). Scale bars as indicated. (H–I) Statistical analysis of NSC morphology (number of filopodia per 1 µm/length of filopodia) after the above described transfection experiments. P -values from ANOVA for multiple-group comparison are

    Article Snippet: The following primary antibodies were used: rabbit anti-SK3 diluted 1∶200, rabbit (Alomone Labs), mouse anti-Nestin monoclonal diluted 1∶500 (BD Biosciences), mouse anti-Abi-1 diluted 1∶250 (MBL), mouse anti-myc antibody diluted 1∶500 (Invitrogen), rabbit anti-PSD95 diluted 1∶1000, rabbit anti-SV2 diluted 1∶300 (both abcam, Cambridge, USA) and rabbit anti-nWASP diluted 1∶500 (Santa Cruz) or chicken anti-nWASP diluted 1∶500 (abcam); fluorescence labeled secondary antibodies were Alexa Fluor® 488 (green, used filter set: excitation BP 450 – 490, FT 510, emission BP 515 - 565), Alexa Fluor® 568 (red, used filter set: excitation BP 534 nm–558 nm, FT 560, emission BP 575 - 640) and Alexa Fluor® 647 (magenta used filter set: excitation BP 610 nm–670 nm, FT 660, emission BP 640–740 (all from Invitrogen) all diluted 1∶500.

    Techniques: Over Expression, Expressing, Transfection, Construct

    Expression of CD140α, CD44, CD34 and SK3 cell surface markers in iMSCs, fibroblasts, and PDGFRα-positive cells. Flow cytometry analysis detected the expression of CD140α (( A ). cell count; ( B) . mean fluorescence intensity), CD44 (( C ). cell count; ( D ). mean fluorescence intensity), CD34 (( E ). cell count; ( F ). mean fluorescence intensity), and SK3 (( G ). cell count; ( H ). mean fluorescence intensity) in iMSCs, fibroblasts, and PDGFRα-positive ( n = 3, * p

    Journal: International Journal of Molecular Sciences

    Article Title: Differentiated PDGFRα-Positive Cells: A Novel In-Vitro Model for Functional Studies of Neuronal Nitric Oxide Synthase

    doi: 10.3390/ijms22073514

    Figure Lengend Snippet: Expression of CD140α, CD44, CD34 and SK3 cell surface markers in iMSCs, fibroblasts, and PDGFRα-positive cells. Flow cytometry analysis detected the expression of CD140α (( A ). cell count; ( B) . mean fluorescence intensity), CD44 (( C ). cell count; ( D ). mean fluorescence intensity), CD34 (( E ). cell count; ( F ). mean fluorescence intensity), and SK3 (( G ). cell count; ( H ). mean fluorescence intensity) in iMSCs, fibroblasts, and PDGFRα-positive ( n = 3, * p

    Article Snippet: Cells were then washed with PBS for 10 min and incubated with the following primary antibodies in a humidified chamber overnight at 4 °C: anti-CD34 (ab81289; Abcam, Cambridge, UK), anti-CD44 (BD Pharmingen, San Diego, CA, USA), anti-KCa2.3-ATTO-594 (SK3; Alomone Labs, Jerusalem, Israel), anti-PDGFRα (ab234965; Abcam, Cambridge, UK), and anti-nNOS (3G6B10; Invitrogen Carlsbad, CA, USA).

    Techniques: Expressing, Flow Cytometry, Cell Counting, Fluorescence