bk channel β4 subunit  (Alomone Labs)

Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: A. Detection of mitoBK Ca channel regulatory β4 subunit mRNA in astrocytoma cells. The BK Ca subunit β4 mRNA was detected at a size of 405 bp. No products were obtained for the BK Ca subunits β1, β2 and β3. GAPDH served as a positive control and was detected at a size of 496 bp. The negative control without reverse transcriptase (−RT) and samples without cDNA (−A) had no signals. The results presented are representative of seven independent experiments. B. Immunoblot of astrocytoma mitochondria, astrocytoma cell homogenate and brain homogenate fractions labeled with the anti-BK Ca channel β4 subunit antibody. A control antigen (BK Ca β4+ peptide) was used as a positive control for the specificity of the antibody. An anti-cytochrome c oxidase subunit IV antibody (COX IV) was used as a mitochondrial marker (n = 3). C. Immuno-gold electron microscopy localization of the BK Ca channel β4 regulatory subunit in mitochondria of cultured human astrocytoma cells. The β4 subunit molecules were labeled using 10 nm colloidal-gold particles (arrows). D. High-power confocal image of cultured astrocytoma cells immunolabeled to detect OxPhos (red) and β4-GFP-transfected cells (green). The superimposition of the two signals revealed the mitochondrial localization of BK Ca β4 in human astrocytoma cells (yellow). The DNA-binding dye DAPI was used to stain the cell nuclei (blue). For details concerning the astrocytoma cells, see the .

Article Snippet: The immunoreactions consisted of sequential incubations with a rabbit polyclonal anti-BK Ca channel β subunit 4 antibody (anti-β4, 1∶20, Alomone Labs) followed by species-specific donkey secondary antibodies coupled to 10 nm gold particles (Electron Microscopy Sciences).

Techniques: Positive Control, Negative Control, Western Blot, Labeling, Marker, Electron Microscopy, Cell Culture, Immunolabeling, Transfection, Binding Assay, Staining

Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: Two-dimensional separation was performed as described in the , and the PVDF membrane was first immunoblotted for the BK Ca channel β4 subunit (below, Coomassie staining panel). Next, the PVDF membrane was immunoblotted for the subunits of individual respiratory chain complexes (below the BK Ca β4 panel). The BN-PAGE was calibrated based on the location of mitochondrial respiratory chain complexes that were isolated from rat heart mitochondria (above the panel for the blue native PAGE of mitochondria from astrocytoma cells). In the native astrocytoma lysate, mitochondria BK Ca β4 co-localized with subunit I of cytochrome c oxidase. M, the monomeric form of cytochrome c oxidase; D, the dimeric form of cytochrome c oxidase; Sc 1 and Sc 2 , complexes with higher molecular weights containing cytochrome c oxidase. A typical immunoblot from three separate experiments is shown.

Article Snippet: The immunoreactions consisted of sequential incubations with a rabbit polyclonal anti-BK Ca channel β subunit 4 antibody (anti-β4, 1∶20, Alomone Labs) followed by species-specific donkey secondary antibodies coupled to 10 nm gold particles (Electron Microscopy Sciences).

Techniques: Staining, Isolation, Blue Native PAGE, Western Blot

Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: A. Detection of mitoBK Ca channel regulatory β4 subunit mRNA in astrocytoma cells. The BK Ca subunit β4 mRNA was detected at a size of 405 bp. No products were obtained for the BK Ca subunits β1, β2 and β3. GAPDH served as a positive control and was detected at a size of 496 bp. The negative control without reverse transcriptase (−RT) and samples without cDNA (−A) had no signals. The results presented are representative of seven independent experiments. B. Immunoblot of astrocytoma mitochondria, astrocytoma cell homogenate and brain homogenate fractions labeled with the anti-BK Ca channel β4 subunit antibody. A control antigen (BK Ca β4+ peptide) was used as a positive control for the specificity of the antibody. An anti-cytochrome c oxidase subunit IV antibody (COX IV) was used as a mitochondrial marker (n = 3). C. Immuno-gold electron microscopy localization of the BK Ca channel β4 regulatory subunit in mitochondria of cultured human astrocytoma cells. The β4 subunit molecules were labeled using 10 nm colloidal-gold particles (arrows). D. High-power confocal image of cultured astrocytoma cells immunolabeled to detect OxPhos (red) and β4-GFP-transfected cells (green). The superimposition of the two signals revealed the mitochondrial localization of BK Ca β4 in human astrocytoma cells (yellow). The DNA-binding dye DAPI was used to stain the cell nuclei (blue). For details concerning the astrocytoma cells, see the .

Article Snippet: The membranes were exposed to polyclonal antibodies that recognize BK Ca channel β subunit 4 (anti-β4, 1∶200, Alomone Labs) and cytochrome c oxidase subunit IV (COX IV, 1∶1000, Cell Signaling).

Techniques: Positive Control, Negative Control, Western Blot, Labeling, Marker, Electron Microscopy, Cell Culture, Immunolabeling, Transfection, Binding Assay, Staining

Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: Two-dimensional separation was performed as described in the , and the PVDF membrane was first immunoblotted for the BK Ca channel β4 subunit (below, Coomassie staining panel). Next, the PVDF membrane was immunoblotted for the subunits of individual respiratory chain complexes (below the BK Ca β4 panel). The BN-PAGE was calibrated based on the location of mitochondrial respiratory chain complexes that were isolated from rat heart mitochondria (above the panel for the blue native PAGE of mitochondria from astrocytoma cells). In the native astrocytoma lysate, mitochondria BK Ca β4 co-localized with subunit I of cytochrome c oxidase. M, the monomeric form of cytochrome c oxidase; D, the dimeric form of cytochrome c oxidase; Sc 1 and Sc 2 , complexes with higher molecular weights containing cytochrome c oxidase. A typical immunoblot from three separate experiments is shown.

Article Snippet: The membranes were exposed to polyclonal antibodies that recognize BK Ca channel β subunit 4 (anti-β4, 1∶200, Alomone Labs) and cytochrome c oxidase subunit IV (COX IV, 1∶1000, Cell Signaling).

Techniques: Staining, Isolation, Blue Native PAGE, Western Blot

Journal: Journal of Applied Physiology

Article Title: Chronic interval exercise training prevents BK Ca channel-mediated coronary vascular dysfunction in aortic-banded miniswine

doi: 10.1152/japplphysiol.01138.2017

Figure Lengend Snippet: In vivo normalized coronary blood flow (CBF) and vascular conductance responses to large-conductance Ca2+-activated K+ (BKCa) channel α-subunit activation in nonsham sedentary control (CON), aortic-banded heart failure sedentary (HF), and aortic-banded heart failure interval exercise-trained (HF-IT) animals. A: CBF response [relative to left ventricle (LV) weight] following BKCa channel α-subunit activation by NS-1619 is dependent on group (repeated-measures ANOVA, P < 0.05). B: exercise training prevents decreased BKCa channel-mediated increases in CBF observed in HF animals, measured as the difference (∆) in CBF following infusion of NS-1619 minus Baseline (1-way ANOVA, P < 0.05). C: NS-1619-induced increases in CBF are attenuated by pretreatment with penitrem A (Pen A) [P = not significant (NS)]. D: increased coronary vascular conductance (CVC; relative to LV weight) following BKCa channel α-subunit activation by NS-1619 is dependent on group (repeated-measures ANOVA, P < 0.05). E: again, exercise training prevents decreased BKCa channel-mediated vasodilatory capacity observed in HF animals, measured as the difference in CVC following infusion of NS-1619 minus Baseline (1-way ANOVA, P < 0.05). F: NS-1619-induced increases in CVC are attenuated by pretreatment with Pen A (P = NS). §Interaction effect: Group × Dose (P < 0.05); *post hoc vs. CON (P < 0.05), †post hoc vs. HF-IT (P < 0.05); #post hoc vs. CON (P < 0.10); ‡post hoc HF vs. HF-IT (P < 0.10). n = 5, 6, and 6 for CON, HF, and HF-IT, respectively.

Article Snippet: Proteins were resolved by SDS-PAGE using 4–20% acrylamide, transferred onto PVDF membranes, and blotted with the following commercially available antibodies: BK Ca channel α-subunit (110–130 kDa, 1:500; NeuroMab) and β-actin (42 kDa, 1:2,500; Sigma-Aldrich).

Techniques: In Vivo, Activation Assay, Control

Journal: Journal of Applied Physiology

Article Title: Chronic interval exercise training prevents BK Ca channel-mediated coronary vascular dysfunction in aortic-banded miniswine

doi: 10.1152/japplphysiol.01138.2017

Figure Lengend Snippet: In vitro coronary arteriole functional responses to large-conductance Ca2+-activated K+ (BKCa) channel α-subunit activation in nonsham sedentary control (CON), aortic-banded heart failure sedentary (HF), and aortic-banded heart failure interval exercise-trained (HF-IT) animals. A: exercise training prevents the decreased vasodilation in isolated coronary arterioles observed in HF animals after activation of the BKCa channel α-subunit by NS-1619 (repeated-measures ANOVA, P < 0.05). B and C: biochemical analysis of isolated coronary arterioles show no differences in BKCa channel α-subunit mRNA levels (B) or protein (C). D: representative Western blots of BKCa channel α-subunit and β-actin (loading control) arteriole protein levels. + CON, positive control – rat brain. A separate group of animals not included in the analysis of the present report were run on the original gel. Since these samples are not relevant to the present study, they have been removed from the gel as indicated by white dividing lines. E: biotinylation analysis reveals no differences in the cellular distribution of the BKCa channel α-subunit in denuded right coronary artery samples. F: representative Western blot of membrane (mem) and cytosolic (cyt) BKCa channel α-subunit protein levels in the right coronary artery. + CON, positive control – CON arteriole sample from D. §Interaction effect: Group × Dose (P < 0.05); *post hoc vs. CON (P < 0.05); †post hoc vs. HF-IT (P < 0.05); #post hoc vs. CON (P < 0.10); ‡post hoc vs. HF-IT (P < 0.10). n = 6, 5, and 6 for CON, HF, and HF-IT, respectively, for A; n = 6, 6, and 7 for CON, HF, and HF-IT, respectively, for B–F.

Article Snippet: Proteins were resolved by SDS-PAGE using 4–20% acrylamide, transferred onto PVDF membranes, and blotted with the following commercially available antibodies: BK Ca channel α-subunit (110–130 kDa, 1:500; NeuroMab) and β-actin (42 kDa, 1:2,500; Sigma-Aldrich).

Techniques: In Vitro, Functional Assay, Activation Assay, Control, Isolation, Western Blot, Positive Control, Membrane