anti bk α subunit antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti bk α subunit antibody
    (A) Representative amplification plots and agarose gel separation of real‐time RT‐PCR analysis of BK β 1‐subunit and GAPDH in MA, colons, and kidneys from WT and BK β 1‐KO mice. The expression threshold was set at 0.22, a level above background fluorescence but within the linear phase of the amplification plot. The intersection between the threshold level and the amplification plot is the Ct value, which correlates with the amount of template in the sample. Ct values over 35 are excluded, as these values approach the sensitivity limits of the Taqman assay. Amplification of real‐time RT‐PCR products was seen at 75 bp in tissues from WT animals only. Con, nontemplate control. (B) Representative western blot obtained using anti‐BK <t>α</t> ‐subunit antibody in colonic and kidney tissues from WT and BK β 1‐KO mice. Antibody detected a protein band at ~100 kDa in all tissues from WT and BK β 1‐KO mice. The signals are blocked by preincubation with the antibody competing peptide (CP). Arrows indicate the manufacturer's recommended molecular weight of BK α ‐subunit protein.
    Anti Bk α Subunit Antibody, 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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    Images

    1) Product Images from "Western blot analysis of BK channel β1‐subunit expression should be interpreted cautiously when using commercially available antibodies"

    Article Title: Western blot analysis of BK channel β1‐subunit expression should be interpreted cautiously when using commercially available antibodies

    Journal: Physiological Reports

    doi: 10.14814/phy2.12189

    (A) Representative amplification plots and agarose gel separation of real‐time RT‐PCR analysis of BK β 1‐subunit and GAPDH in MA, colons, and kidneys from WT and BK β 1‐KO mice. The expression threshold was set at 0.22, a level above background fluorescence but within the linear phase of the amplification plot. The intersection between the threshold level and the amplification plot is the Ct value, which correlates with the amount of template in the sample. Ct values over 35 are excluded, as these values approach the sensitivity limits of the Taqman assay. Amplification of real‐time RT‐PCR products was seen at 75 bp in tissues from WT animals only. Con, nontemplate control. (B) Representative western blot obtained using anti‐BK α ‐subunit antibody in colonic and kidney tissues from WT and BK β 1‐KO mice. Antibody detected a protein band at ~100 kDa in all tissues from WT and BK β 1‐KO mice. The signals are blocked by preincubation with the antibody competing peptide (CP). Arrows indicate the manufacturer's recommended molecular weight of BK α ‐subunit protein.
    Figure Legend Snippet: (A) Representative amplification plots and agarose gel separation of real‐time RT‐PCR analysis of BK β 1‐subunit and GAPDH in MA, colons, and kidneys from WT and BK β 1‐KO mice. The expression threshold was set at 0.22, a level above background fluorescence but within the linear phase of the amplification plot. The intersection between the threshold level and the amplification plot is the Ct value, which correlates with the amount of template in the sample. Ct values over 35 are excluded, as these values approach the sensitivity limits of the Taqman assay. Amplification of real‐time RT‐PCR products was seen at 75 bp in tissues from WT animals only. Con, nontemplate control. (B) Representative western blot obtained using anti‐BK α ‐subunit antibody in colonic and kidney tissues from WT and BK β 1‐KO mice. Antibody detected a protein band at ~100 kDa in all tissues from WT and BK β 1‐KO mice. The signals are blocked by preincubation with the antibody competing peptide (CP). Arrows indicate the manufacturer's recommended molecular weight of BK α ‐subunit protein.

    Techniques Used: Amplification, Agarose Gel Electrophoresis, Quantitative RT-PCR, Mouse Assay, Expressing, Fluorescence, TaqMan Assay, Western Blot, Molecular Weight

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    Alomone Labs mouse anti bk channel α
    Membrane current in  HEK  293 cells stably expressing human  BK  channel α‐ and β1‐subunits.  (A)  Voltage‐dependent  BK  current traces recorded in a representative cell with the voltage protocol as shown in the  inset  before and after application of 1 μM paxilline (a selective  BK  channel blocker), and washout of paxilline.  (B)  I‐V  relationships of  BK  current in the absence and presence of 1 μM paxilline, and upon washout ( n  = 5, * P 
    Mouse Anti Bk Channel α, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Alomone Labs kcnmb4
    BK Ca openers control spasticity. (A) RNAseq expression of BK Ca channel components in EA.hy926 cells. Inset represents the western blot of <t>KCNMB4</t> in EA.hy926 cells, which was tested in four samples, and western blot of KCNMB4 expression in the spinal cord in control ( n = 3) and spastic EAE spinal cord tissues (two shown, n = 6 total) showing an additional prominent band, not present in EA.hy926 cells, above the anticipated fragment size more notable during EAE. This antibody detected multiple bands in western blot analyses. (B) RNAseq expression of BK Ca channel components in the spinal cords of spastic EAE ( n = 3 mice) and age‐matched normal mice ( n = 3 mice). The results represent the mean ± SD. Inset represents the western blot of KCNMA1 in control and spastic EAE tissue (Antibody APC‐021 detected a single band at the anticipated size and was repeated with antibody APC‐151 with comparable results), and insets show a significant change in ratio between Kcnma1 and neurofilament light ( Nefl ) gene levels. (C) Percentage change in resistance to flexion of spastic hindlimbs in ABH mice in response to treatment with 5 mg·kg −1 p.o. VSN16R in water ( n = 10 animals, n = 19 limbs) that were pretreated with saline or 1 mg·kg −1 i.p. paxilline ( n = 7 animals, n = 14 limbs). (D) Percentage change in resistance to flexion of hindlimbs in ABH mice following injection i.p. with 1 mg·kg −1 paxilline ( n = 7 animals, n = 13 limbs), 40 mg·kg −1 i.p. BMS‐204352 ( n = 7 animals, n = 13 limbs), 20 mg·kg −1 i.p. NS‐1619 ( n = 7 animals, n = 14 limbs) or 10 mg·kg −1 i.p. NS‐11021 ( n = 7 animals, n = 13 limbs). *Significant difference compared with baseline.
    Kcnmb4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Alomone Labs anti bkα
    BK channels and NMDARs interact via <t>BKα</t> and GluN1 in HEK-293 cells. ( A ) Reciprocal pull-down of GluN1 and BKα by anti-BKα and -GluN1 antibodies in the absence of expression of any other NMDAR subunit. IB, immunoblot; IP, immunopurification. ( B ) Pull-down of GluN2A and GluN2B by an anti-BKα antibody only in the presence of GluN1 expression. ( C ) Pull-down of BKα by an anti-GluN2A or -GluN2B antibody only in the presence of GluN1 expression. ( D ) Reciprocal pull-down assay showed that the BKα was largely defective in its association with GluN1 upon deletion of its S0–S1 loop region (residues 46–93). ( E ) Pull-down of BKα by an anti-GluN1 was interfered by a synthesized peptide of the BKα’s S0–S1 loop region (residues 46–93), but not by a scrambled peptide. The peptides were added at a concentration of 1 mg/mL to cell lysates that were equally divided from the same lysate of cells coexpressing BKα and GluN1. ( F ) Pull-down of GluN1 by an anti-BKα antibody showed that the BKα–GluN1 association was markedly decreased with the GluN1 N1/K2 C mutant in which the C-terminal part, including TM domain (residues 527–647 and 781–826) and C-terminal domain (residues 827–920), was replaced by those of GluK2. ( G ) Pull-down of the GluN1’s cytosolic regions by the BKα46–93 peptide, but not by the scrambled peptide. The peptides were biotinylated on their N termini and immobilized on streptavidin agarose. The fusion construct of the GluN1’s cytosolic regions (residues 563–587 and 813–920) was 6× His-tagged in its C terminus, expressed in E. coli , and purified with IMAC chromatography.
    Anti Bkα, 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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    Membrane current in  HEK  293 cells stably expressing human  BK  channel α‐ and β1‐subunits.  (A)  Voltage‐dependent  BK  current traces recorded in a representative cell with the voltage protocol as shown in the  inset  before and after application of 1 μM paxilline (a selective  BK  channel blocker), and washout of paxilline.  (B)  I‐V  relationships of  BK  current in the absence and presence of 1 μM paxilline, and upon washout ( n  = 5, * P 

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Tyrphostin AG556 increases the activity of large conductance Ca2+‐activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase

    doi: 10.1111/jcmm.13103

    Figure Lengend Snippet: Membrane current in HEK 293 cells stably expressing human BK channel α‐ and β1‐subunits. (A) Voltage‐dependent BK current traces recorded in a representative cell with the voltage protocol as shown in the inset before and after application of 1 μM paxilline (a selective BK channel blocker), and washout of paxilline. (B) I‐V relationships of BK current in the absence and presence of 1 μM paxilline, and upon washout ( n  = 5, * P 

    Article Snippet: Proteins were immunoprecipitated overnight at 4°C using 1 μg of mouse anti‐BK channel α (APC‐021; Alomone Labs, Jerusalem, Israel) antibody or 1 μg of mouse anti‐β1 (APC‐036; Alomone Labs, Jerusalem, Israel) antibody and 20 μl of Protein A/G beads (sc‐2003; Santa Cruz Biotechnology, Inc. CA, USA).

    Techniques: Stable Transfection, Expressing

    Depolymerization of microtubules decreases the number of BKα and RyR2 colocalization sites. ( A ) Wide-field image of a freshly isolated arterial myocyte immunolabeled for BKα ( n = 12 cells, n = 3 animals). Red box indicates the area where superresolution images were obtained. Scale bar, 10 μm. ( B ) Superresolution localization map obtained after immunolabeling with anti-BKμ (green) and anti-RyR2 (red) antibodies ( n = 12 cells, n = 3 animals). Scale bar, 3 μm. ROIs (yellow boxes) are shown in magnified view (I) and (II) below. Scale bars, 0.2 μm. ( C ) Cluster size distribution histograms of RyR2 and BKα ( n = 11,005 or n = 11,940 particles for RyR2 and BKα, respectively; 12 cells, n = 3 animals). ( D ) Summary of object-based analysis to determine the number of BKα protein cluster centroids per cell that overlay with RyR2 protein clusters in control cells, cells in which a random BKα distribution has been simulated, and cells treated with nocodazole (10 μM). * P ≤ 0.05 compared to control ( n =12 cells per group, 3 animals). Coloc., colocalization.

    Journal: Science signaling

    Article Title: Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility

    doi: 10.1126/scisignal.aan2694

    Figure Lengend Snippet: Depolymerization of microtubules decreases the number of BKα and RyR2 colocalization sites. ( A ) Wide-field image of a freshly isolated arterial myocyte immunolabeled for BKα ( n = 12 cells, n = 3 animals). Red box indicates the area where superresolution images were obtained. Scale bar, 10 μm. ( B ) Superresolution localization map obtained after immunolabeling with anti-BKμ (green) and anti-RyR2 (red) antibodies ( n = 12 cells, n = 3 animals). Scale bar, 3 μm. ROIs (yellow boxes) are shown in magnified view (I) and (II) below. Scale bars, 0.2 μm. ( C ) Cluster size distribution histograms of RyR2 and BKα ( n = 11,005 or n = 11,940 particles for RyR2 and BKα, respectively; 12 cells, n = 3 animals). ( D ) Summary of object-based analysis to determine the number of BKα protein cluster centroids per cell that overlay with RyR2 protein clusters in control cells, cells in which a random BKα distribution has been simulated, and cells treated with nocodazole (10 μM). * P ≤ 0.05 compared to control ( n =12 cells per group, 3 animals). Coloc., colocalization.

    Article Snippet: Cells were fixed with 3.2% formaldehyde/0.1% glutaraldehyde–phosphate-buffered saline (PBS), permeabilized and blocked with 0.2% saponin/5% horse serum–PBS, and incubated with primary antibodies against α-tubulin (1:100; MA1-80017, Life Technologies), BKα (1:100; APC-021, Alomone Labs), and RyR2 (1:100; ab2868, Abcam).

    Techniques: Isolation, Immunolabeling

    BK Ca openers control spasticity. (A) RNAseq expression of BK Ca channel components in EA.hy926 cells. Inset represents the western blot of KCNMB4 in EA.hy926 cells, which was tested in four samples, and western blot of KCNMB4 expression in the spinal cord in control ( n = 3) and spastic EAE spinal cord tissues (two shown, n = 6 total) showing an additional prominent band, not present in EA.hy926 cells, above the anticipated fragment size more notable during EAE. This antibody detected multiple bands in western blot analyses. (B) RNAseq expression of BK Ca channel components in the spinal cords of spastic EAE ( n = 3 mice) and age‐matched normal mice ( n = 3 mice). The results represent the mean ± SD. Inset represents the western blot of KCNMA1 in control and spastic EAE tissue (Antibody APC‐021 detected a single band at the anticipated size and was repeated with antibody APC‐151 with comparable results), and insets show a significant change in ratio between Kcnma1 and neurofilament light ( Nefl ) gene levels. (C) Percentage change in resistance to flexion of spastic hindlimbs in ABH mice in response to treatment with 5 mg·kg −1 p.o. VSN16R in water ( n = 10 animals, n = 19 limbs) that were pretreated with saline or 1 mg·kg −1 i.p. paxilline ( n = 7 animals, n = 14 limbs). (D) Percentage change in resistance to flexion of hindlimbs in ABH mice following injection i.p. with 1 mg·kg −1 paxilline ( n = 7 animals, n = 13 limbs), 40 mg·kg −1 i.p. BMS‐204352 ( n = 7 animals, n = 13 limbs), 20 mg·kg −1 i.p. NS‐1619 ( n = 7 animals, n = 14 limbs) or 10 mg·kg −1 i.p. NS‐11021 ( n = 7 animals, n = 13 limbs). *Significant difference compared with baseline.

    Journal: British Journal of Pharmacology

    Article Title: Big conductance calcium‐activated potassium channel openers control spasticity without sedation) Big conductance calcium‐activated potassium channel openers control spasticity without sedation

    doi: 10.1111/bph.13889

    Figure Lengend Snippet: BK Ca openers control spasticity. (A) RNAseq expression of BK Ca channel components in EA.hy926 cells. Inset represents the western blot of KCNMB4 in EA.hy926 cells, which was tested in four samples, and western blot of KCNMB4 expression in the spinal cord in control ( n = 3) and spastic EAE spinal cord tissues (two shown, n = 6 total) showing an additional prominent band, not present in EA.hy926 cells, above the anticipated fragment size more notable during EAE. This antibody detected multiple bands in western blot analyses. (B) RNAseq expression of BK Ca channel components in the spinal cords of spastic EAE ( n = 3 mice) and age‐matched normal mice ( n = 3 mice). The results represent the mean ± SD. Inset represents the western blot of KCNMA1 in control and spastic EAE tissue (Antibody APC‐021 detected a single band at the anticipated size and was repeated with antibody APC‐151 with comparable results), and insets show a significant change in ratio between Kcnma1 and neurofilament light ( Nefl ) gene levels. (C) Percentage change in resistance to flexion of spastic hindlimbs in ABH mice in response to treatment with 5 mg·kg −1 p.o. VSN16R in water ( n = 10 animals, n = 19 limbs) that were pretreated with saline or 1 mg·kg −1 i.p. paxilline ( n = 7 animals, n = 14 limbs). (D) Percentage change in resistance to flexion of hindlimbs in ABH mice following injection i.p. with 1 mg·kg −1 paxilline ( n = 7 animals, n = 13 limbs), 40 mg·kg −1 i.p. BMS‐204352 ( n = 7 animals, n = 13 limbs), 20 mg·kg −1 i.p. NS‐1619 ( n = 7 animals, n = 14 limbs) or 10 mg·kg −1 i.p. NS‐11021 ( n = 7 animals, n = 13 limbs). *Significant difference compared with baseline.

    Article Snippet: Membranes were washed, blocked for 1 h in blocking buffer (5% non‐fat dried milk) and incubated in blocking buffer with primary antibody using rabbit polyclonal antibodies against mouse/human KCNMA1 (APC‐021, previously validated by lack of activity in big conductance calcium‐activated potassium channel Kcnma1 ‐deficient mice and APC‐151 antibody) and KCNMB4 (APC‐061 antibody, previously validated by lack of activity in Kcnmb4‐deficient mice despite detecting multiple isoforms), which were purchased from Alomone Labs, Jerusalem Israel, whose website reports supporting literature concerning characterization of the antibodies.

    Techniques: Expressing, Western Blot, Mouse Assay, Injection

    BK channels and NMDARs interact via BKα and GluN1 in HEK-293 cells. ( A ) Reciprocal pull-down of GluN1 and BKα by anti-BKα and -GluN1 antibodies in the absence of expression of any other NMDAR subunit. IB, immunoblot; IP, immunopurification. ( B ) Pull-down of GluN2A and GluN2B by an anti-BKα antibody only in the presence of GluN1 expression. ( C ) Pull-down of BKα by an anti-GluN2A or -GluN2B antibody only in the presence of GluN1 expression. ( D ) Reciprocal pull-down assay showed that the BKα was largely defective in its association with GluN1 upon deletion of its S0–S1 loop region (residues 46–93). ( E ) Pull-down of BKα by an anti-GluN1 was interfered by a synthesized peptide of the BKα’s S0–S1 loop region (residues 46–93), but not by a scrambled peptide. The peptides were added at a concentration of 1 mg/mL to cell lysates that were equally divided from the same lysate of cells coexpressing BKα and GluN1. ( F ) Pull-down of GluN1 by an anti-BKα antibody showed that the BKα–GluN1 association was markedly decreased with the GluN1 N1/K2 C mutant in which the C-terminal part, including TM domain (residues 527–647 and 781–826) and C-terminal domain (residues 827–920), was replaced by those of GluK2. ( G ) Pull-down of the GluN1’s cytosolic regions by the BKα46–93 peptide, but not by the scrambled peptide. The peptides were biotinylated on their N termini and immobilized on streptavidin agarose. The fusion construct of the GluN1’s cytosolic regions (residues 563–587 and 813–920) was 6× His-tagged in its C terminus, expressed in E. coli , and purified with IMAC chromatography.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Glutamate-activated BK channel complexes formed with NMDA receptors

    doi: 10.1073/pnas.1802567115

    Figure Lengend Snippet: BK channels and NMDARs interact via BKα and GluN1 in HEK-293 cells. ( A ) Reciprocal pull-down of GluN1 and BKα by anti-BKα and -GluN1 antibodies in the absence of expression of any other NMDAR subunit. IB, immunoblot; IP, immunopurification. ( B ) Pull-down of GluN2A and GluN2B by an anti-BKα antibody only in the presence of GluN1 expression. ( C ) Pull-down of BKα by an anti-GluN2A or -GluN2B antibody only in the presence of GluN1 expression. ( D ) Reciprocal pull-down assay showed that the BKα was largely defective in its association with GluN1 upon deletion of its S0–S1 loop region (residues 46–93). ( E ) Pull-down of BKα by an anti-GluN1 was interfered by a synthesized peptide of the BKα’s S0–S1 loop region (residues 46–93), but not by a scrambled peptide. The peptides were added at a concentration of 1 mg/mL to cell lysates that were equally divided from the same lysate of cells coexpressing BKα and GluN1. ( F ) Pull-down of GluN1 by an anti-BKα antibody showed that the BKα–GluN1 association was markedly decreased with the GluN1 N1/K2 C mutant in which the C-terminal part, including TM domain (residues 527–647 and 781–826) and C-terminal domain (residues 827–920), was replaced by those of GluK2. ( G ) Pull-down of the GluN1’s cytosolic regions by the BKα46–93 peptide, but not by the scrambled peptide. The peptides were biotinylated on their N termini and immobilized on streptavidin agarose. The fusion construct of the GluN1’s cytosolic regions (residues 563–587 and 813–920) was 6× His-tagged in its C terminus, expressed in E. coli , and purified with IMAC chromatography.

    Article Snippet: Rabbit polyclonal anti-GluN1 (catalog no. G8913; Sigma-Aldrich), anti-BKα (catalog no. APC-107; Alomone Labs), anti-GluN2A (catalog no. AB1555; EMD Millipore), anti-GluN2B (catalog no. SAB2104208; Sigma-Aldrich), and anti-FLAG (catalog no. F7425; Sigma-Aldrich) and mouse monoclonal anti-BKα (catalog no. L6/60; NeuroMabs) antibodies were used for immunopurification.

    Techniques: Expressing, Immu-Puri, Pull Down Assay, Synthesized, Concentration Assay, Mutagenesis, Construct, Purification, Chromatography

    BK channels and NMDARs form complexes in rat brains. ( A ) Schematic of the sample-preparation protocols for the immunopurified NMDAR (IP-NMDAR) and BK channel (IP-BK), tandem immunopurified BK–NMDAR complex (TIP-BK/NMDAR), and negative control samples. ( B ) SDS/PAGE and silver stain analysis of the IP-NMDAR and control samples. ( C ) Tandem mass spectrometric spectra of a unique peptide of BKα identified in the IP-NMDAR sample. ( D ) Immunoblot (IB) analysis of BKα and GluN1 in the IP-NMDAR sample. ( E ) Tandem mass spectrometric spectra for a unique peptide of GluN1 identified in the IP-BK sample. ( F ) Immunoblot analysis of GluN1 and BKα in the IP-BK sample. ( G ) Immunoblot analysis of GluN2A and GluN2B in the TIP-BK/NMDAR sample. ( H ) Immunoblot analysis of BKα and GluN1 in IP-NMDAR ( Left ) and IP-BK ( Right ) samples prepared from different brain regions.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Glutamate-activated BK channel complexes formed with NMDA receptors

    doi: 10.1073/pnas.1802567115

    Figure Lengend Snippet: BK channels and NMDARs form complexes in rat brains. ( A ) Schematic of the sample-preparation protocols for the immunopurified NMDAR (IP-NMDAR) and BK channel (IP-BK), tandem immunopurified BK–NMDAR complex (TIP-BK/NMDAR), and negative control samples. ( B ) SDS/PAGE and silver stain analysis of the IP-NMDAR and control samples. ( C ) Tandem mass spectrometric spectra of a unique peptide of BKα identified in the IP-NMDAR sample. ( D ) Immunoblot (IB) analysis of BKα and GluN1 in the IP-NMDAR sample. ( E ) Tandem mass spectrometric spectra for a unique peptide of GluN1 identified in the IP-BK sample. ( F ) Immunoblot analysis of GluN1 and BKα in the IP-BK sample. ( G ) Immunoblot analysis of GluN2A and GluN2B in the TIP-BK/NMDAR sample. ( H ) Immunoblot analysis of BKα and GluN1 in IP-NMDAR ( Left ) and IP-BK ( Right ) samples prepared from different brain regions.

    Article Snippet: Rabbit polyclonal anti-GluN1 (catalog no. G8913; Sigma-Aldrich), anti-BKα (catalog no. APC-107; Alomone Labs), anti-GluN2A (catalog no. AB1555; EMD Millipore), anti-GluN2B (catalog no. SAB2104208; Sigma-Aldrich), and anti-FLAG (catalog no. F7425; Sigma-Aldrich) and mouse monoclonal anti-BKα (catalog no. L6/60; NeuroMabs) antibodies were used for immunopurification.

    Techniques: Sample Prep, Negative Control, SDS Page, Silver Staining

    In situ PLA of BKα and GluN1 colocalization in HEK-293 cells and mouse brains. ( A ) PLA signals (red dots) probed with anti-V5 and -FLAG antibodies under a nonpermeabilized condition were detected in HEK-293 cells coexpressing V5-BKα and FLAG-GluN1. ( B and C ) In situ PLA of BKα–GluN1 complexes in the hippocampal dentate gyrus of control (Nestin-Cre − /KCNMA1 fl/fl ) mice ( B ) and neuron-specific BKα KO (Nestin-Cre + /KCNMA1 fl/fl ) mice ( C ). Nuclei are shown in blue (DAPI). (Scale bars: 40 or 10 μm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Glutamate-activated BK channel complexes formed with NMDA receptors

    doi: 10.1073/pnas.1802567115

    Figure Lengend Snippet: In situ PLA of BKα and GluN1 colocalization in HEK-293 cells and mouse brains. ( A ) PLA signals (red dots) probed with anti-V5 and -FLAG antibodies under a nonpermeabilized condition were detected in HEK-293 cells coexpressing V5-BKα and FLAG-GluN1. ( B and C ) In situ PLA of BKα–GluN1 complexes in the hippocampal dentate gyrus of control (Nestin-Cre − /KCNMA1 fl/fl ) mice ( B ) and neuron-specific BKα KO (Nestin-Cre + /KCNMA1 fl/fl ) mice ( C ). Nuclei are shown in blue (DAPI). (Scale bars: 40 or 10 μm.)

    Article Snippet: Rabbit polyclonal anti-GluN1 (catalog no. G8913; Sigma-Aldrich), anti-BKα (catalog no. APC-107; Alomone Labs), anti-GluN2A (catalog no. AB1555; EMD Millipore), anti-GluN2B (catalog no. SAB2104208; Sigma-Aldrich), and anti-FLAG (catalog no. F7425; Sigma-Aldrich) and mouse monoclonal anti-BKα (catalog no. L6/60; NeuroMabs) antibodies were used for immunopurification.

    Techniques: In Situ, Proximity Ligation Assay, Mouse Assay

    Postsynaptic BK channels regulate synaptic transmission in mature dentate granule cells via NMDAR-mediated channel activation. ( A ) Effects of paxilline (Pax) alone and combined with AP5 on the amplitudes of evoked EPSPs ( n = 13). ( B ) Effects of AP5 alone and combined with paxilline on the amplitudes of evoked EPSPs ( n = 13). For comparison, the effects of AP5 alone on evoked EPSPs over a similar extended time course ( n = 6) were included in the averaged plot. ( C ) Effects of paxilline alone and combined with AP5 on the amplitudes of evoked EPSPs in mature granule cells in BKα-KO (Nestin-Cre + /KCNMA1 fl/fl ) mice ( n = 9). ( D ) The effect of paxilline alone (bath) and combined with AP5 (bath) on evoked EPSPs in the presence of intracellularly applied MK-801 ( n = 9). ( E ) Effects of paxilline on evoked EPSPs in the presence of intracellularly applied paxilline ( n = 8). ( F ) Effects of paxilline on evoked EPSPs in the presence of intracellularly applied 0.5 mg/mL BKα46–93 ( n = 8) or scrambled ( n = 7) peptide. Pep., peptide. ( G ) Effects of extracellularly and intracellularly applied paxilline ( n = 9 and 7, respectively) and KO of BK channels (Nestin-Cre + /KCNMA1 fl/fl ) ( n = 5) on paired-pulse ratios. All experiments were done with regular C57BL/C6 mice except as specified. Control, data obtained without or before drug application. Paxilline and AP5 were perfused in the bath solution at a concentration of 10 and 200 µM, respectively, and they were applied either individually or combined together at a later stage of the experiment. Statistical differences were evaluated by using a t test. N.S., not significant. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Glutamate-activated BK channel complexes formed with NMDA receptors

    doi: 10.1073/pnas.1802567115

    Figure Lengend Snippet: Postsynaptic BK channels regulate synaptic transmission in mature dentate granule cells via NMDAR-mediated channel activation. ( A ) Effects of paxilline (Pax) alone and combined with AP5 on the amplitudes of evoked EPSPs ( n = 13). ( B ) Effects of AP5 alone and combined with paxilline on the amplitudes of evoked EPSPs ( n = 13). For comparison, the effects of AP5 alone on evoked EPSPs over a similar extended time course ( n = 6) were included in the averaged plot. ( C ) Effects of paxilline alone and combined with AP5 on the amplitudes of evoked EPSPs in mature granule cells in BKα-KO (Nestin-Cre + /KCNMA1 fl/fl ) mice ( n = 9). ( D ) The effect of paxilline alone (bath) and combined with AP5 (bath) on evoked EPSPs in the presence of intracellularly applied MK-801 ( n = 9). ( E ) Effects of paxilline on evoked EPSPs in the presence of intracellularly applied paxilline ( n = 8). ( F ) Effects of paxilline on evoked EPSPs in the presence of intracellularly applied 0.5 mg/mL BKα46–93 ( n = 8) or scrambled ( n = 7) peptide. Pep., peptide. ( G ) Effects of extracellularly and intracellularly applied paxilline ( n = 9 and 7, respectively) and KO of BK channels (Nestin-Cre + /KCNMA1 fl/fl ) ( n = 5) on paired-pulse ratios. All experiments were done with regular C57BL/C6 mice except as specified. Control, data obtained without or before drug application. Paxilline and AP5 were perfused in the bath solution at a concentration of 10 and 200 µM, respectively, and they were applied either individually or combined together at a later stage of the experiment. Statistical differences were evaluated by using a t test. N.S., not significant. * P

    Article Snippet: Rabbit polyclonal anti-GluN1 (catalog no. G8913; Sigma-Aldrich), anti-BKα (catalog no. APC-107; Alomone Labs), anti-GluN2A (catalog no. AB1555; EMD Millipore), anti-GluN2B (catalog no. SAB2104208; Sigma-Aldrich), and anti-FLAG (catalog no. F7425; Sigma-Aldrich) and mouse monoclonal anti-BKα (catalog no. L6/60; NeuroMabs) antibodies were used for immunopurification.

    Techniques: Transmission Assay, Activation Assay, Mouse Assay, Concentration Assay