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: 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: Low Salt Delivery Triggers Autocrine Release of Prostaglandin E2 From the Aldosterone-Sensitive Distal Nephron in Familial Hyperkalemic Hypertension Mice

doi: 10.3389/fphys.2021.787323

Figure Lengend Snippet: Antibody list.

Article Snippet: Bk-beta1 , Alomone APC-036 , Rabbit , WB 1:6,000.

Techniques: Immunofluorescence, Western Blot

Journal: Frontiers in Physiology

Article Title: Low Salt Delivery Triggers Autocrine Release of Prostaglandin E2 From the Aldosterone-Sensitive Distal Nephron in Familial Hyperkalemic Hypertension Mice

doi: 10.3389/fphys.2021.787323

Figure Lengend Snippet: Inhibition of the EP1 receptor rescues renal outer medullary potassium (ROMK) protein abundance. (A) plasma K+, (B) Total K+ excretion: total K+ intake ratio per individual mouse, (C) transtubular K+ gradient of an individual mouse, and (D) western blot and analysis of ROMK and (E) BK-beta1. * p < 0.05; statistical significance evaluated by two-tailed t -test.

Article Snippet: Bk-beta1 , Alomone APC-036 , Rabbit , WB 1:6,000.

Techniques: Inhibition, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: MiR-339-3p aggravates rat vascular inflammation induced by AT1R autoantibodies by down-regulating BKα protein expression

doi: 10.1101/2021.10.17.464722

Figure Lengend Snippet: (A) 12 weeks after active immunization with AT1R-ECII, the OD value of serum AT1-AA in rats was detected. Detection of (B) caudal vein blood pressure and (C) vascular wall thickness of thoracic aorta in AT1-AA-positive rats, n=5. (D) The light and heavy chains of AT1-AA isolated by SDS-PAGE gel. (E) Effect of AT1-AA on the beating of neonatal rat cardiomyocytes. (F) The effect of AT1-AA on vasomotion was detected by vascular ring. Detection of BKβ1 protein level in (G) thoracic aorta of AT1-AA-positive rats and (I) VSMCs treated with AT1-AA, n=5. (H) Detection of BKβ1 protein level in thoracic aorta of AT1-AA-positive rats by immunofluorescence method, n=4.

Article Snippet: After the sections were deparaffinized and hydrated, the antigen was repaired by the high-pressure method, blocked with 10% goat serum, and then incubated with primary antibodies against BKα (Alomone Labs, APC-107, Israel), BKβ1 (Alomone Labs, APC-036, Israel), CD3 (Abcam, ab5690, UK), CD19 (Bioss, bs-0079R, China), CD68 (Affinity Biosciences., DF7518, USA) and α-SMA (Abcam, ab7817, UK) at 4°C overnight.

Techniques: Isolation, SDS Page, Immunofluorescence

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca 2+ -activated K + channels in bronchial smooth muscle cells

doi: 10.1152/ajplung.00331.2019

Figure Lengend Snippet: Large-conductance Ca2+-activated K+ channel γ1-subunit (BKγ1) expression is selectively high in mouse bronchial smooth muscle (BSM). A: real-time PCR analyses of BKγ subunits (BKγ1–4) in mouse bronchial smooth muscles (BSMs; N = 3). B: real-time PCR analyses of BKα, BKβ1, and BKγ1 in several types of mouse SM tissues (N = 4–5). C: real-time PCR analysis was performed to compare BKγ1 mRNA expression between mouse trachea and bronchus (trachea, N = 3; bronchus, N = 5). *P < 0.05 vs. trachea by Student’s t test. D: Western blotting analyses of BKα, BKβ1, BKγ1, and β-actin, using specific antibodies, respectively, in mouse aortic and bronchial SMs (mASMs and mBSMs, respectively) on the same membrane. Similar results were obtained from 3 independent experiments. To obtain enough protein, 3 mice were used for each experiment. E: immunocytostaining was performed to reveal cellular localization of BKα and BKγ1. Images were acquired using a confocal microscope. Merged fluorescent images were overlapped with a cell image (right). Aortic SMC was used as a control, in which BKγ1 expression is low.

Article Snippet: The blots were incubated with anti-LRRC26 antibody (1:100 dilution, sc-132325; Santa Cruz Biotechnology, Houston, TX), anti-BKα antibody (1:200 dilution, APC-107, Alomone Laboratories), or anti-BKβ1 antibody (1:100 dilution, APC-036, Alomone Laboratories) and then incubated with horseradish peroxidase-conjugated IgG antibody (Chemicon International, Temecula, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Microscopy

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca 2+ -activated K + channels in bronchial smooth muscle cells

doi: 10.1152/ajplung.00331.2019

Figure Lengend Snippet: Voltage-dependence of the large-conductance Ca2+-activated K+ (BK) channels in mouse bronchial smooth muscle cells (mBSMCs), mouse aortic SMCs (mASMCs), and a HEK293-based heterologous system. A: BK currents were recorded at pCa 8.0 using whole cell patch-clamp recordings in HEK293 cells expressing BKα-mCherry (BKα), BKα-mCherry + BKβ1-GFP (BKα+β1), BKα+γ1, and BKα + BKβ1-mCherry + BKγ1-GFP (BKα+β1+γ1). K+ currents were recorded in response to pulse protocols shown in insets. Representative traces of whole cell currents and tail currents from each cell are shown. B: BK activation curves in mBSMCs (n = 5), mASMCs (n = 5), BKα (n = 5), BKα+β1 (n = 5), BKα+γ1 (n = 5), and BKα+β1+γ1 (n = 4) in A and Fig. 4. Relationships between selected test potential by the maximum current amplitude (G/Gmax) and voltage were fitted with the Boltzmann equation. C: voltage values required for half-maximum activation (V1/2) were calculated by the activation curve of B. *P < 0.05 vs. mBSMCs, by Tukey’s test.

Article Snippet: The blots were incubated with anti-LRRC26 antibody (1:100 dilution, sc-132325; Santa Cruz Biotechnology, Houston, TX), anti-BKα antibody (1:200 dilution, APC-107, Alomone Laboratories), or anti-BKβ1 antibody (1:100 dilution, APC-036, Alomone Laboratories) and then incubated with horseradish peroxidase-conjugated IgG antibody (Chemicon International, Temecula, CA).

Techniques: Patch Clamp, Expressing, Activation Assay