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: 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: 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 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: 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

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: Aging (Albany NY)

Article Title: BK channel overexpression on plasma membrane of fibroblasts from Hutchinson-Gilford progeria syndrome

doi: 10.18632/aging.101621

Figure Lengend Snippet: Immunofluorescence detection for BK Ca channels expressed on plasma membrane in fixed cells. ( A ) Fluorescence micrographs of isolated hDF obtained from young and HGPS donors incubated with an anti-BK Ca α subunit primary antibody visualized by FITC-conjugated secondary antibody and acquired at 200× magnification. Scale bars: 100 μm. ( B ). Quantification of mean fluorescence intensity of anti-BK Ca antibody-stained cells. The green fluorescence intensity values are obtained from 30 cells (A.U. ± SEM). Significant differences calculated according to the Student’s t-test (p<0.05) are indicated.

Article Snippet: The BK Ca α subunits were labeled by using the rabbit polyclonal IgG specific to the extracellular amino acids residues KCNMA1-199:213 (Alomone Labs, Jerusalem, Israel) diluted 1:50 in PBS and incubated overnight at 4°C.

Techniques: Immunofluorescence, Fluorescence, Isolation, Incubation, Staining

Journal: Aging (Albany NY)

Article Title: BK channel overexpression on plasma membrane of fibroblasts from Hutchinson-Gilford progeria syndrome

doi: 10.18632/aging.101621

Figure Lengend Snippet: Immunofluorescence detection for BK Ca channels expressed on plasma membrane in living cells. ( A ) Fluorescence and fluorescence/phase contrast merged micrographs of isolated hDF obtained from young, elderly and HGPS donors incubated with an anti-BK Ca α subunit primary antibody visualized by the conjugated Alexa Fluor 350 fluorophore and acquired at 200× magnification. Scale bars: 100 μm. ( B ) Histogram showing the percentage of cells expressing a blue fluorescence intensity over a fixed threshold (% ± SEM). Significant differences calculated according to the Student’s t-test (p<0.05) are indicated.

Article Snippet: The BK Ca α subunits were labeled by using the rabbit polyclonal IgG specific to the extracellular amino acids residues KCNMA1-199:213 (Alomone Labs, Jerusalem, Israel) diluted 1:50 in PBS and incubated overnight at 4°C.

Techniques: Immunofluorescence, Fluorescence, Isolation, Incubation, Expressing

Journal: Aging (Albany NY)

Article Title: BK channel overexpression on plasma membrane of fibroblasts from Hutchinson-Gilford progeria syndrome

doi: 10.18632/aging.101621

Figure Lengend Snippet: Proliferation and adhesion rates without and with the BK Ca inhibitor IbTx. ( A ) Average number of hDF obtained from young healthy donors and patients affected by HGPS estimated at 48, 72 and 96 hours from seeding and normalized at 24 h (fold-change ± SEM). Healthy and HGPS hDF were allowed to proliferate untreated (Young n=54; HGPS n=36) and treated by 100 nM IbTx (Young n=53; HGPS n=36). Young vs. HGPS: **p<0.001; ****p<5×10 -7 ; Young vs. Young + IbTx: §p<0.01; HGPS vs. HGPS + IbTx: #p<0.01; ###p<0.0001. ( B ) Average percentage ± SEM of adherent hDF cells counted 2 h after seeding, treated (Young n=35, HGPS n=22) and untreated by 100 nM IbTx (Young n=40, HGPS n=24); image legend is the same as Figure A.

Article Snippet: The BK Ca α subunits were labeled by using the rabbit polyclonal IgG specific to the extracellular amino acids residues KCNMA1-199:213 (Alomone Labs, Jerusalem, Israel) diluted 1:50 in PBS and incubated overnight at 4°C.

Techniques: