rabbit polyclonal anti bk ca channel β subunit 4 antibody  (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: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Aldosterone treatment increases the frequency and the amplitude of STOCs in MASMCs without modifying BK channel subunit expression. (A) Representative traces of STOCs recorded at a holding potential of –40 mV from MASMCs in the absence (CONTROL, black trace ) or after 24 h-treatment with aldosterone 10 nM (ALDO, red trace ). Scatterplots with mean ± SEM illustrate STOC frequency ( B , normalized with respect to cell capacitance, in events/s/pF), STOC amplitude ( C , normalized with respect to cell capacitance, in pA/pF), and STOC area-under-the-curve ( D , in pA.s) in control MASMCs ( n = 119 events/ n = 12 cells/ N = 4 animals, empty circles ) and ALDO-treated cells ( n = 333 events/ n = 12 cells/ N = 5 animals, red circles ). * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control group. (E) Histogram distribution of normalized STOC amplitudes in control ( n = 119 events/ n = 12 cells/ N = 4 rats, white bars ) and ALDO-treated MASMCs ( n = 333 events/ n = 12 cells/ N = 5 rats, red bars ) indicates the increase in the amplitude of STOCs above 3.6 pA/pF in ALDO-treated cells. (F,G) Representative immunoblot images and scatterplot with mean ± SEM of BK Ca channel α subunit expression ( n = 4 control samples, empty circles ; n = 4 ALDO-treated samples, red circles ), and β1 subunit expression ( n = 5 control samples, empty triangles ; n = 5 ALDO-treated samples, red triangles ). Each sample was prepared with a pool of 3–4 MA segments from three rats. Values were normalized with respect to GAPDH expression.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Proposed model for ALDO-mediated upregulation of the functional unit that controls Ca 2+ dynamics at the SR-PM nanodomain of MASMCs. In normal conditions (CONTROL, left ) K + -mediated membrane depolarization induces a Ca 2+ influx via Ca v 1.2 (LTCCs). This Ca 2+ entry is buffered by the SERCA pump toward the luminal SR Ca 2+ reservoirs activating RyRs (Ca 2+ sparks) and BK Ca channels (STOCs). Ca v 1.2, SERCA pump, RyRs and BK Ca channels work as a functional unit in the PM-SR nanodomain regulating [Ca 2+ ] cyt , luminal SR Ca 2+ levels, and opposing vasoconstriction. The treatment of MAs with aldosterone (ALDO, right ) increases Ca v 1.2 protein expression and induces higher Ca 2+ entry in MASMCs. However, the depolarization-induced vascular contraction was not enhanced because of the upregulation of this functional unit, which involves increased expression and activity of SERCA pump controlling abnormal Ca 2+ influx at the PM-SR nanodomain, increasing SR Ca 2+ content, Ca 2+ spark and STOC frequencies, opposing to depolarization-induced vasoconstriction and enhancing ACh-mediated vasorelaxation.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Functional Assay, Expressing, Activity Assay

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Aldosterone treatment increases Ca v 1.2 expression but does not change depolarization-induced vasoconstriction responses of rat mesenteric arteries. (A) Representative traces of tension changes ( top recordings ) in response to potassium chloride (KCl) additions (from 10 to 60 mM KCl) of rat MA rings treated with aldosterone (ALDO 10 nM, 24 h, red trace ) or without it (Control, black trace ). The concentration-response graph ( below ) shows vasoconstriction responses of MA rings treated ( red circles ) or not ( empty circles ) with ALDO (10 nM, 24 h). Depolarization-induced contraction was expressed as a percentage of maximal contractile response induced by 60 mM KCl. Data are shown as mean ± SEM of n = 7 MA rings from N = 7 rats for each experimental group. (B) Representative immunoblot image and scatterplot with mean ± SEM ( below ) of Ca v 1.2 protein expression from homogenates of control MAs ( empty circles ), ALDO-treated MAs ( red circles ), and ALDO-treated MAs co-incubated with RU 28318, a selective MR antagonist (1 μM, red/black circles ). Data are shown as mean ± SEM of n = 6 independent experiments for each group. Each experiment was performed with a pool of 3–4 MA segments from three rats. * P < 0.01 vs. untreated MAs. # P < 0.05 vs. ALDO-treated MAs.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Expressing, Control, Concentration Assay, Western Blot, Incubation

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: SERCA pump counterbalances depolarization-induced Ca 2+ entry in ALDO-treated MASMCs. (A) Representative Ca 2+ influx recordings ( F / F 0 ) of Fluo 4-loaded MASMCs from control ( empty circles ) or ALDO-treated ( red circles ) cells preincubated with thapsigargin (TGN) 100 nM for 10 min ( white/black and red/black circles , respectively) to block SERCA pump activity, or in its absence. Cells were kept in Na + and Ca 2+ free solution. After 2-min of frame-scan recording, cells were perfused with Na + free solution (to block Na + /Ca 2+ exchanger activity) containing 1.8 mM CaCl 2 plus 20 mM KCl to induce LTCC-mediated Ca 2+ influx. Changes in cytoplasmic Ca 2+ were recorded with a laser scanning confocal microscope (Zeiss, LSM 700) equipped with an ×63 oil immersion objective (N.A. 1.2). (B) Scatterplot with mean ± SEM illustrates the amplitude of depolarization-induced Ca 2+ entry (Δ F / F 0 ) of Fluo 4-loaded MASMCs under the different experimental conditions. The amplitude of Ca 2+ influx was determined at minute 8 of the recording in control MASMCs ( n = 17 cells/ N = 4 rats, empty circles ), ALDO-treated MASMCs ( n = 13 cells/ N = 5 rats, empty circles ), control MASMCs + TGN ( n = 15 cells/ N = 6 rats, white/black circles ) and ALDO-treated MASMCs + TGN ( n = 13 cells/ N = 5 rats, red/black circles ), respectively. *** P < 0.001 vs. control cells; ### P < 0.001 vs. ALDO-treated cells; &&& P < 0.001 vs. control + TGN cells. (C) Representative immunoblot image and scatterplot with mean ± SEM ( below ) of SERCA pump protein expression from homogenates of control MAs ( empty circles ), ALDO-treated MAs ( red circles ), and ALDO-treated MAs co-incubated with 1 μM RU28318, a selective MR antagonist ( red/black circles ). Data are shown as mean ± SEM of n = 5 experiments for each group. Each experiment was performed with a pool of 3–4 MA segments from three rats. SERCA pump expression levels were normalized to the expression of GAPDH for each independent experiment. * P < 0.05 vs. control group. # P < 0.05 vs. ALDO-treated group. (D) Scatterplot with mean ± SEM of Atp2a2 relative mRNA levels determined by real-time qPCR from control MAs ( empty circles , n = 8), ALDO-treated MAs ( red circles , n = 8), and ALDO-treated MAs co-incubated with 1 μM RU28318 ( red/black circles, n = 3). * P < 0.05 vs. control group.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Control, Blocking Assay, Activity Assay, Microscopy, Western Blot, Expressing, Incubation

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Aldosterone treatment increases Ca 2+ spark frequency in MASMCs. (A) Representative pseudo-colored confocal images of Ca 2+ sparks ( top ) and normalized ( F / F 0 ) fluorescence profiles ( bottom ) from Fluo 4-loaded MASMCs treated or not with aldosterone (ALDO 10 nM, 24 h) and preincubated or not with Nifedipine (1 μM, 10 min). The fluorescence profile was calculated in the region indicated by the green bar. (B) Scatterplot with mean ± SEM illustrates Ca 2+ spark frequency in Fluo 4-loaded MASMCs from control ( empty circles , n = 35 cells/ N = 5 rats); ALDO-treated arteries ( red circles , n = 29 cells/ N = 4 rats); pre-incubated with Nifedipine (NIF, white/black circles, n = 18 cells/ N = 4 rats; and red/black circles, n = 11 cells/ N = 3 rats, respectively). ** P < 0.01 vs. control cells. # P < 0.05 and ### P < 0.001 vs. ALDO-treated group.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Fluorescence, Control, Incubation

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Aldosterone treatment increases the frequency and the amplitude of STOCs in MASMCs without modifying BK channel subunit expression. (A) Representative traces of STOCs recorded at a holding potential of –40 mV from MASMCs in the absence (CONTROL, black trace ) or after 24 h-treatment with aldosterone 10 nM (ALDO, red trace ). Scatterplots with mean ± SEM illustrate STOC frequency ( B , normalized with respect to cell capacitance, in events/s/pF), STOC amplitude ( C , normalized with respect to cell capacitance, in pA/pF), and STOC area-under-the-curve ( D , in pA.s) in control MASMCs ( n = 119 events/ n = 12 cells/ N = 4 animals, empty circles ) and ALDO-treated cells ( n = 333 events/ n = 12 cells/ N = 5 animals, red circles ). * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control group. (E) Histogram distribution of normalized STOC amplitudes in control ( n = 119 events/ n = 12 cells/ N = 4 rats, white bars ) and ALDO-treated MASMCs ( n = 333 events/ n = 12 cells/ N = 5 rats, red bars ) indicates the increase in the amplitude of STOCs above 3.6 pA/pF in ALDO-treated cells. (F,G) Representative immunoblot images and scatterplot with mean ± SEM of BK Ca channel α subunit expression ( n = 4 control samples, empty circles ; n = 4 ALDO-treated samples, red circles ), and β1 subunit expression ( n = 5 control samples, empty triangles ; n = 5 ALDO-treated samples, red triangles ). Each sample was prepared with a pool of 3–4 MA segments from three rats. Values were normalized with respect to GAPDH expression.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Expressing, Control, Western Blot

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Enhanced acetylcholine-mediated vasorelaxation of ALDO-treated MAs. Relaxation responses of MAs treated (ALDO, red circles ) or not (CONTROL, empty circles ) with aldosterone (10 nM, 24 h) to increasing concentrations of acetylcholine (ACh, from 0.001 to 1 μM). MA rings were pre-contracted with KCl 60 mM and exposed to cumulative ACh concentrations ( n = 6 MA rings/ N = 6 rats, for each experimental condition). Relaxation is shown as a percentage with respect to the contraction response produced by 60 mM KCl, which was taken as 100%. Data are shown as mean ± SEM. * P < 0.05 vs. non-linear data fit of control arteries.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Control, Produced

Journal: Frontiers in Physiology

Article Title: Aldosterone-Induced Sarco/Endoplasmic Reticulum Ca 2+ Pump Upregulation Counterbalances Ca v 1.2-Mediated Ca 2+ Influx in Mesenteric Arteries

doi: 10.3389/fphys.2022.834220

Figure Lengend Snippet: Proposed model for ALDO-mediated upregulation of the functional unit that controls Ca 2+ dynamics at the SR-PM nanodomain of MASMCs. In normal conditions (CONTROL, left ) K + -mediated membrane depolarization induces a Ca 2+ influx via Ca v 1.2 (LTCCs). This Ca 2+ entry is buffered by the SERCA pump toward the luminal SR Ca 2+ reservoirs activating RyRs (Ca 2+ sparks) and BK Ca channels (STOCs). Ca v 1.2, SERCA pump, RyRs and BK Ca channels work as a functional unit in the PM-SR nanodomain regulating [Ca 2+ ] cyt , luminal SR Ca 2+ levels, and opposing vasoconstriction. The treatment of MAs with aldosterone (ALDO, right ) increases Ca v 1.2 protein expression and induces higher Ca 2+ entry in MASMCs. However, the depolarization-induced vascular contraction was not enhanced because of the upregulation of this functional unit, which involves increased expression and activity of SERCA pump controlling abnormal Ca 2+ influx at the PM-SR nanodomain, increasing SR Ca 2+ content, Ca 2+ spark and STOC frequencies, opposing to depolarization-induced vasoconstriction and enhancing ACh-mediated vasorelaxation.

Article Snippet: Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Ca v 1.2 (1:200, Cat# AB10515, Millipore, Merck KGaA, Darmstadt, Germany) ( ); SERCA2 pump (1:4,000, Cat# ab2861, Abcam, Cambridge, MA, United States) ( ); Ryanodine receptor (RyR, 1:5000, Cat# ab2868, Abcam, Cambridge, MA, United States) ( ); calsequestrin (CSQ2, 1:4,000, Cat# ab108289, Abcam, Cambridge, MA, United States) ( ); sorcin (1:1,000, a kind gift from Héctor H. Valdivia laboratory, University of Wisconsin, Madison, WI, United States) ( ); FKBP12.6 (1:2,000, Cat# sc-376135, Santa Cruz Biotechnology, Inc., Dallas, TX, United States) ( ); MR (1:200; Cat# MRN2 2B7, DSHB, University of Iowa, Iowa City, IA, United States) ( ); BK Ca α subunit (1:200, Cat# APC-009, Alomone Labs, Jerusalem, Israel) , BK Ca β1 subunit (1:5000, Cat# APC-036, Alomone Labs, Jerusalem, Israel) , Orai1 (1:200, Cat# O8264, Sigma–Aldrich Química, S.L.

Techniques: Functional Assay, Control, Membrane, Expressing, Activity Assay

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Sequence of primers used for RT-PCR

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Sequencing

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Decreased BK Ca currents of ACC pyramidal neurons from mCCD rats. a , Voltage ranges from − 60 to + 40 mV with 10 mV increments, typical recordings of BK Ca currents in pyramidal neurons from sham (left) and mCCD (right) rats, BK Ca currents were isolated with paxilline (10 μM). b , The I-V relationship curves showed the differences in the ACC pyramidal neurons from mCCD ( n = 12) and sham (n = 12) rats. c , BK Ca current density at + 40 mV from voltage-clamp recordings of ACC pyramidal neurons from mCCD (n = 12) and sham (n = 12) rats. ** p < 0.01 compared with that of sham group

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Isolation

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Upregulation of BK Ca β4 subunit protein and mRNA in the ACC during neuropathic pain associated anxiety-like behavior. a, The schematic diagram of the behavioral and biochemical experiments. b, Quantification of the mRNA levels of BK Ca channel subunits in the ACC between mCCD (n = 7) and sham ( n = 6) rats on postoperative day 7. c, Quantification of the mRNA levels of BK Ca channels in the insular cortex between mCCD (n = 7) and sham (n = 6) rats on postoperative day 7. d, Quantification of the mRNA levels of BK Ca β4 subunit in the ACC between mCCD (n = 6) and sham (n = 6) rats on postsurgical day 3, 7, 14. e, Representative Western blots for BK Ca α and BK Ca β4 subunit in the ACC obtained on postsurgical day 7. f, Quantification of the protein levels of α and β4 subunits in the ACC between mCCD (n = 3) and sham (n = 3) rats on postoperative day 7. * p < 0.05, ** p < 0.01 compared with that of sham group

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Western Blot

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Postsynaptic and presynaptic upregulation of BK Ca β4 subunit protein in the ACC during neuropathic pain associated anxiety-like behavior. a , Representative Western blots for PSD 95, syntaxin 1A, BK Ca β4 and BK Ca α subunits in the non-PSD and PSD membrane fractions of the ACC in sham and mCCD rats on postoperative day 14; b , BK Ca β4 subunit was significantly enhanced in the PSD and non-PSD fractions of the ACC from mCCD (PSD fraction: 164 ± 37%, n = 6, * p < 0.05; non-PSD fraction: 224 ± 32%, n = 6, * p < 0.05) rats on postoperative day 14 compared with those in sham rats (PSD fraction: 100 ± 32%, n = 6; non-PSD fraction: 100 ± 12%, n = 6). BK Ca α subunit showed no significant change in the PSD fraction between sham (100 ± 17%, n = 6) and mCCD (91.4 ± 14%, n = 6, p > 0.05) rats on postoperative day 14

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Western Blot

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Effects of activating BK Ca channels in the ACC on neuropathic pain and anxiety-like behaviors. a, A schematic diagram of microinjection and the behavioral experiment. b, Bilateral microinjection of NS1619 (10 μM, 0.5 μL) into the ACC reversed the time in the open arms and the open arm entries in mCCD rats on day 7 after surgery. c, Rats infused with NS1619 spent more time in the central areas in open field test in mCCD rats compared with ACSF-treated animals. There was no significant difference in the total distance travelled within the open field for 15 min among mCCD and sham rats (sham + ACSF: n = 6, sham +NS1619: n = 6, mCCD + ACSF: n = 6, mCCD +NS1619: n = 6). d, The pain threshold of the injured hind paw (left) and contralateral feet (right), after bilateral microinjection of NS1619 into the ACC on day 7 after surgery. # p < 0.05, ## p < 0.01 compared with that of mCCD with ACSF group

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques:

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Sequence of primers used for RT-PCR

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Sequencing

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Decreased BK Ca currents of ACC pyramidal neurons from mCCD rats. a , Voltage ranges from − 60 to + 40 mV with 10 mV increments, typical recordings of BK Ca currents in pyramidal neurons from sham (left) and mCCD (right) rats, BK Ca currents were isolated with paxilline (10 μM). b , The I-V relationship curves showed the differences in the ACC pyramidal neurons from mCCD ( n = 12) and sham (n = 12) rats. c , BK Ca current density at + 40 mV from voltage-clamp recordings of ACC pyramidal neurons from mCCD (n = 12) and sham (n = 12) rats. ** p < 0.01 compared with that of sham group

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Isolation

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Upregulation of BK Ca β4 subunit protein and mRNA in the ACC during neuropathic pain associated anxiety-like behavior. a, The schematic diagram of the behavioral and biochemical experiments. b, Quantification of the mRNA levels of BK Ca channel subunits in the ACC between mCCD (n = 7) and sham ( n = 6) rats on postoperative day 7. c, Quantification of the mRNA levels of BK Ca channels in the insular cortex between mCCD (n = 7) and sham (n = 6) rats on postoperative day 7. d, Quantification of the mRNA levels of BK Ca β4 subunit in the ACC between mCCD (n = 6) and sham (n = 6) rats on postsurgical day 3, 7, 14. e, Representative Western blots for BK Ca α and BK Ca β4 subunit in the ACC obtained on postsurgical day 7. f, Quantification of the protein levels of α and β4 subunits in the ACC between mCCD (n = 3) and sham (n = 3) rats on postoperative day 7. * p < 0.05, ** p < 0.01 compared with that of sham group

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Western Blot

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Postsynaptic and presynaptic upregulation of BK Ca β4 subunit protein in the ACC during neuropathic pain associated anxiety-like behavior. a , Representative Western blots for PSD 95, syntaxin 1A, BK Ca β4 and BK Ca α subunits in the non-PSD and PSD membrane fractions of the ACC in sham and mCCD rats on postoperative day 14; b , BK Ca β4 subunit was significantly enhanced in the PSD and non-PSD fractions of the ACC from mCCD (PSD fraction: 164 ± 37%, n = 6, * p < 0.05; non-PSD fraction: 224 ± 32%, n = 6, * p < 0.05) rats on postoperative day 14 compared with those in sham rats (PSD fraction: 100 ± 32%, n = 6; non-PSD fraction: 100 ± 12%, n = 6). BK Ca α subunit showed no significant change in the PSD fraction between sham (100 ± 17%, n = 6) and mCCD (91.4 ± 14%, n = 6, p > 0.05) rats on postoperative day 14

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Western Blot, Membrane

Journal: Molecular Brain

Article Title: Upregulation of Beta4 subunit of BK Ca channels in the anterior cingulate cortex contributes to mechanical allodynia associated anxiety-like behaviors

doi: 10.1186/s13041-020-0555-z

Figure Lengend Snippet: Effects of activating BK Ca channels in the ACC on neuropathic pain and anxiety-like behaviors. a, A schematic diagram of microinjection and the behavioral experiment. b, Bilateral microinjection of NS1619 (10 μM, 0.5 μL) into the ACC reversed the time in the open arms and the open arm entries in mCCD rats on day 7 after surgery. c, Rats infused with NS1619 spent more time in the central areas in open field test in mCCD rats compared with ACSF-treated animals. There was no significant difference in the total distance travelled within the open field for 15 min among mCCD and sham rats (sham + ACSF: n = 6, sham +NS1619: n = 6, mCCD + ACSF: n = 6, mCCD +NS1619: n = 6). d, The pain threshold of the injured hind paw (left) and contralateral feet (right), after bilateral microinjection of NS1619 into the ACC on day 7 after surgery. # p < 0.05, ## p < 0.01 compared with that of mCCD with ACSF group

Article Snippet: Membranes were incubated with primary antibody against BK Ca α subunits (1:400, Alomone Labs, Jerusalem, Israel; product no., APC-021), BK Ca β4 subunit (1:200, Alomone Labs, Jerusalem, Israel; product no., APC-061) or β-actin (1:1000, Sigma) as loading control overnight at 4 °C.

Techniques: Microinjection

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

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

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, Isolation, Western Blot, Positive 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 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: Vessels that spontaneously developed tone in this range were subsequently exposed to seven incremental logarithmic doses of the BK Ca channel α-subunit agonist NS-1619 (Sigma-Aldrich, St. Louis, MO) ranging from 1e −10 to 1e −4 .

Techniques: In Vivo, Activation Assay

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: Vessels that spontaneously developed tone in this range were subsequently exposed to seven incremental logarithmic doses of the BK Ca channel α-subunit agonist NS-1619 (Sigma-Aldrich, St. Louis, MO) ranging from 1e −10 to 1e −4 .

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