Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Effects of inhibition of AGEs on coronary artery tensions and BK channel densities and protein expression (a) Representative tracings for 60 mmol/L KCl and 100 nmol/L IBTX induced vascular tension alterations of coronary arterial rings from C+V, DM+V, C+A and DM+A groups. (b) Graph data showing the vascular tension alterations induced by KCl. (c) Graph data showing the vascular tension alterations (IBTX/KCl). (d and e) Whole-cell potassium currents before and after application of 100 nmol/L IBTX, and the I-V relationship of IBTX-sensitive currents of control and AGEs-cultured freshly isolated rat coronary arterial SMCs ( n = 3∼6 per group). (f) The representative tracings of baseline potassium currents and potassium currents after application of 100 nM IBTX in rat coronary arterial SMCs of the C+V, DM+V, C+A and DM+A groups, respectively ( n = 3∼5 per group). (g) Graph data showing IBTX-sensitive current densities at the testing potential of +100 mV in rat coronary arterial SMCs of the four groups. (h–j) The protein expressions of BK-α and BK-β1 in human coronary arterial SMCs in the BSA and BSA-AGEs groups ( n = 6∼9 per group). Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels. (k-l) The mRNA expression of BK-α and BK-β1 in rat coronary arteries of the C+V, DM+V, C+A and DM+A groups. β-actin was used as an internal control to normalize differences in the amount of total RNA in each rat sample ( n = 4 per group). (m and n) The mRNA expression of BK-α and BK-β1 in human coronary arterial SMCs of the NG, HG, NG+A, HG+A groups. GAPDH was used as an internal control to normalize differences in the amount of total RNA in each cell sample ( n = 4∼5 per group). (o–q) Protein expressions of BK-α and BK-β1 in rat coronary arteries of the C+V, DM+V, C+A and DM+A groups. Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels ( n = 5 per group). (r–t) Protein expressions of BK-α and BK-β1 in human coronary arterial SMCs of the NG, HG, NG+A, HG+A groups. Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels ( n = 5∼9 per group). (C+V: Control + Vehicle; C+A: Control + aminoguanidine; DM+V: DM + Vehicle; DM+A: DM + aminoguanidine. NG: normal glucose; HG: high glucose; NG+A: normal glucose + aminoguanidine; HG+A: high glucose + aminoguanidine).

Article Snippet: The PVDF membranes were then incubated with specific primary antibodies, including GAPDH (Cell Signaling Technology Co., #2118), BK-α (Santa Cruz Biotechnology, #sc-374,142), BK-β1 (Alomone Labs, #APC-036), anti-AMPK antibody (Cell Signaling Technology, #2532), anti-p-AMPK antibody (Cell Signaling Technology, #2535), anti-FBXO32 antibody (Abcam, #ab194791), anti-Akt antibody (Cell Signaling Technology, #4691), and anti-p-Akt antibody (Cell Signaling Technology, #4060).

Techniques: Inhibition, Expressing, Cell Culture, Isolation

Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Regulation of Akt in AGEs-mediated FBXO32-induced BK-β1 degradation (a and b) Protein expression of FBXO32 in rat coronary arteries of four groups ( n = 5 per group). (c and d) Protein expression of FBXO32 in human coronary arterial SMCs of four cell groups. Quantitative analysis of FBXO32 was normalized to GAPDH protein expression levels. (e–g) Phosphorylation levels of Akt and total Akt in rat coronary arteries of four groups ( n = 8 per group). (h–j) Phosphorylation levels of Akt and total Akt in human coronary arterial SMCs of four groups ( n = 3 per group). The phosphorylation level of Akt (k and n) and the protein expressions of FBXO32 (l and o) and BK-β1 (m and p) were measured after human coronary arterial SMCs were incubated for 96 h in DMEM containing 25.5 mmol/L glucose, or 25.5 mmol/L glucose with aminoguanidine in the absence or presence of MK2206 (0.3 μM) ( n = 5∼10 per group). MK2206 was added at the beginning and remained for 6 h (C+V: Control + Vehicle; C+A: Control + aminoguanidine; DM+V: DM + Vehicle; DM+A: DM + aminoguanidine. NG: normal glucose; HG: high glucose; NG+A: normal glucose + aminoguanidine; HG+A: high glucose + aminoguanidine.)

Article Snippet: The PVDF membranes were then incubated with specific primary antibodies, including GAPDH (Cell Signaling Technology Co., #2118), BK-α (Santa Cruz Biotechnology, #sc-374,142), BK-β1 (Alomone Labs, #APC-036), anti-AMPK antibody (Cell Signaling Technology, #2532), anti-p-AMPK antibody (Cell Signaling Technology, #2535), anti-FBXO32 antibody (Abcam, #ab194791), anti-Akt antibody (Cell Signaling Technology, #4691), and anti-p-Akt antibody (Cell Signaling Technology, #4060).

Techniques: Expressing, Incubation

Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Regulation of AMPK in Akt-mediated FBXO32-induced BK-β1 degradation by AGEs (a–c) Protein expression of p-AMPK and AMPK in rat coronary arteries from the four groups ( n = 8 per group). (d–f) Protein expression of p-AMPK and AMPK in human coronary arterial SMCs from the four groups ( n = 9 per group). Quantitative analysis of p-AMPK and AMPK was normalized to GAPDH protein expression levels. (g) Human coronary arterial SMCs were incubated for 96 h in DMEM containing 25.5 mmol/L glucose, or 25.5 mmol/L glucose and aminoguanidine in the absence or presence of Compound C (CC, 1 μM). Subsequently, the phosphorylation level of AMPK (h and i), AKT (j and k), and the protein expressions of FBXO32 (l) and BK-β1 (m) were measured ( n = 8 and 9 per group). Quantitative analysis of FBXO32 and BK-β1 was normalized to GAPDH protein expression levels.

Article Snippet: The PVDF membranes were then incubated with specific primary antibodies, including GAPDH (Cell Signaling Technology Co., #2118), BK-α (Santa Cruz Biotechnology, #sc-374,142), BK-β1 (Alomone Labs, #APC-036), anti-AMPK antibody (Cell Signaling Technology, #2532), anti-p-AMPK antibody (Cell Signaling Technology, #2535), anti-FBXO32 antibody (Abcam, #ab194791), anti-Akt antibody (Cell Signaling Technology, #4691), and anti-p-Akt antibody (Cell Signaling Technology, #4060).

Techniques: Expressing, Incubation

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: BK Ca -β1 expression is reduced in the carotid arteries of rat balloon-injured model. Representative images of H&E (A) and immunohistochemical staining of BK Ca −α and BK ca −β1 (B) in injured and sham-operated carotid arteries of rat balloon-injured model at 14 days post injury. Scale bar = 100 μm. (C) Quantified mRNA levels of BK ca −α and BK ca −β1 analyzed by qRT-PCR. Western blot images (D) and quantification (E) for indicated proteins. Eukaryotic initiation factor 5 (eIF5) was used as an internal control. Data are presented as means ± SEM ( n = 4∼6 for each group, ns, not significant, * P < 0.05, ** P < 0.01 vs. sham).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: BK Ca -β1 expression is positively associated with VSMC differentiation markers in vitro . Western blot images (A) and quantification (B) of BK ca −α, BK ca −β1, and VSMC differentiation markers expression in VSMCs of passage 1 and passage 6. Western blot images (C) and quantification (D) of BK ca −α, BK ca −β1, and VSMC differentiation markers expression in VSMCs after PDGF-BB treatment (25 μg/L). Western blot images (E) and quantification (F) of BK ca −α, BK ca −β1, and VSMC differentiation markers expression in VSMCs treated with TGF-β (2.5 μg/L). VSMCs were subjected to serum-starvation for 24 h and then treated with the indicated stimulation for 48 h. Passages 3-5 of VSMCs were used in (C) through (F) . Eukaryotic initiation factor 5 (eIF5) was used as an internal control. Data are presented as means ± SEM ( n = 5 for each group, ns, not significant, * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Expressing, In Vitro, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: Decreased BK ca −β1 expression is mediated by elevated PDGF-BB in vivo . (A) Quantified mRNA levels of PDGF-B and TGF-β analyzed by qRT-PCR in injured and sham-operated carotid arteries. Western blot images (B) and quantification (C) of BK ca −α and BK ca −β1 in the carotid arteries of mice injected with saline or PDGF-BB via tail vein. Data are presented as means ± SEM ( n = 6 for each group, ns, not significant, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Expressing, In Vivo, Quantitative RT-PCR, Western Blot, Injection

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: BK Ca -β1 is essential for VSMCs to maintain a differentiated phenotype. (A) Representative micrographs of VSMCs treated with BK ca −β1 siRNA or scramble siRNA transfection. (B) Representative immunofluorescent images of F-actin of VSMCs stained with phalloidin (red). Nuclei were stained with DAPI (blue). Scale bar = 25 μm. (C) The percentage of spindle-like or polygonal-like cells in each group. Western blot images (D) and quantification (E) of BK ca −β1, α-SMA, and SM22α. Representative images (F) and quantification (G) of the areas of collagen gel in each group. The size of the collagen gel in each well was calculated compared to the total area of the well. Data are presented as means ± SEM ( n = 5∼6 for each group, *** P < 0.001 vs. scramble).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Transfection, Staining, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: BK Ca -β1 knockdown induces proliferative, migratory, and synthetic phenotype of VSMCs. (A) Proliferation curves of VSMCs transfected with BK ca −β1 or scrambled siRNA. Representative images (B) and quantification (C) of migration assay. (D) Quantified mRNA levels of inflammatory factors analyzed by qRT-PCR. Representative images (E) and quantification (F) of activities of MMP-9 and MMP-2 tested using gelatin zymography. Data are presented as means ± SEM ( n = 5∼6 for each group, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. scramble).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Transfection, Migration, Quantitative RT-PCR, Zymography

Journal: Frontiers in Cardiovascular Medicine

Article Title: Large-conductance Ca 2 + -activated K + channel β1-subunit maintains the contractile phenotype of vascular smooth muscle cells

doi: 10.3389/fcvm.2022.1062695

Figure Lengend Snippet: Decreased BK ca −β1 expression is correlated with VSMC dedifferentiation and atherosclerosis in human. Western blot images (A) and quantification (B) of BK ca −α and BK ca −β1 expression in human primary aortic VSMCs after PDGF-BB treatment (25 μg/L). Western blot images (C) and quantification (D) of BK ca −α and BK ca −β1 expression in human primary aortic VSMCs treated with TGF-β (2.5 μg/L). VSMCs were subjected to serum-starvation for 24 h and then treated with the indicated stimulation for 48 h. Passages 3–5 of VSMCs were used. (E) Representative western blot images of the protein levels of BK ca −α and BK ca −β1 in lysates of human carotid endarterectomy artery (CEA) and control internal mammary arteries (IMA). (F) Quantification was performed by calculating the ratio between the levels of BK ca −β1 and BK ca −α. Eukaryotic initiation factor 5 (eIF5) was used as an internal control. Data are presented as means ± SEM (n = 3∼5 for each group, ns, not significant, * P < 0.05, ** P < 0.01 vs. control).

Article Snippet: An antibody against BK Ca -β1 (APC-036) used for immunohistochemical staining and western blot was purchased from Alomone labs (Jerusalem, Israel).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Abnormal Ca 2+ Spark/STOC Coupling in Cerebral Artery Smooth Muscle Cells of Obese Type 2 Diabetic Mice

doi: 10.1371/journal.pone.0053321

Figure Lengend Snippet: A . Representative Western Blots of BK α and β1 subunits obtained from control and db/db vascular tissues. Samples containing 30 µg of protein from aorta whole homogenates were run on 15% acrylamide gels and probed with anti-BK α subunit antibody (1∶1000), anti-BK β1 subunit antibody (1∶500) and anti-actin (1∶8000). B . Bar graph represents normalized BKβ1/α densitometric ratios (n = 7 for each group). * P <0.05.

Article Snippet: Supernatants were fractionated on 15% SDS-PAGE gels, transferred onto nitrocellulose membranes (1 h at 100 V, Hybond-ECL, GE Healthcare Bio-Sciences Corp, NJ, USA) and probed with anti-BK α antibody (1∶1000; Alomone Labs, Jerusalem Israel), anti BK β1 antibody (1∶500; Alomone Labs, Jerusalem Israel), anti RyR (1∶5000; Thermo Scientific) and anti-actin antibody (1∶8000, Sigma-Aldrich) in phosphate-buffered saline solution containing Tween-20 (PBS-T; in mmol/L: 3 KH 2 PO 4 , 10 Na 2 HPO 4 , 150 NaCl, 0.1% Tween-20, pH 7.2–7.4).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Abnormal Ca 2+ Spark/STOC Coupling in Cerebral Artery Smooth Muscle Cells of Obese Type 2 Diabetic Mice

doi: 10.1371/journal.pone.0053321

Figure Lengend Snippet: A . Representative Western Blots of BK α and β1 subunits obtained from control and db/db vascular tissues. Samples containing 30 µg of protein from aorta whole homogenates were run on 15% acrylamide gels and probed with anti-BK α subunit antibody (1∶1000), anti-BK β1 subunit antibody (1∶500) and anti-actin (1∶8000). B . Bar graph represents normalized BKβ1/α densitometric ratios (n = 7 for each group). * P <0.05.

Article Snippet: Supernatants were fractionated on 15% SDS-PAGE gels, transferred onto nitrocellulose membranes (1 h at 100 V, Hybond-ECL, GE Healthcare Bio-Sciences Corp, NJ, USA) and probed with anti-BK α antibody (1∶1000; Alomone Labs, Jerusalem Israel), anti BK β1 antibody (1∶500; Alomone Labs, Jerusalem Israel), anti RyR (1∶5000; Thermo Scientific) and anti-actin antibody (1∶8000, Sigma-Aldrich) in phosphate-buffered saline solution containing Tween-20 (PBS-T; in mmol/L: 3 KH 2 PO 4 , 10 Na 2 HPO 4 , 150 NaCl, 0.1% Tween-20, pH 7.2–7.4).

Techniques: 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