immunoblot  (Alomone Labs)

Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: SK Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells

doi: 10.1161/HYPERTENSIONAHA.116.07094

Figure Lengend Snippet: Effect of knockdown of SK2 channels on aldosterone secretion in H295R cells (A) The mRNA expression of SK2 channels was reduced by 70% in H295R cells transduced with shRNA-SK2 lentiviruses. (B) knockdown of SK2 channels increased basal aldosterone secretion, and abrogated apamin-induced increase. *, P

Article Snippet: Slices were blocked (10% horse serum, 0.3% Triton X-100 in PBS, 2h, RT), and then incubated with primary antibody: rabbit anti-KCNN2 (1:200, Alomone Labs), 1% horse serum, 0.3% Triton X-100 in PBS, 1 day, 4°C.

Techniques: Expressing, Transduction, shRNA

Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: SK Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells

doi: 10.1161/HYPERTENSIONAHA.116.07094

Figure Lengend Snippet: H295R cells express functional SK channels (A-C) representative K + current traces in the absence and presence of SK channel activator 1-EBIO, or inhibitor apamin. (D) Apamin-sensitive SK channel currents obtained by subtracting C from B. (E) Current-voltage relationship ( I–V curves) generated from peak current density at each test voltage. Data are presented as means ± S.E.M. from 10 cells. (F) SK1, SK2 and SK3 channel mRNA expression were detected in H295R cells by RT-PCR. NTC: no template control.

Article Snippet: Slices were blocked (10% horse serum, 0.3% Triton X-100 in PBS, 2h, RT), and then incubated with primary antibody: rabbit anti-KCNN2 (1:200, Alomone Labs), 1% horse serum, 0.3% Triton X-100 in PBS, 1 day, 4°C.

Techniques: Functional Assay, Generated, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Neurophysiology

Article Title: Increasing SK2 channel activity impairs associative learning

doi: 10.1152/jn.00025.2012

Figure Lengend Snippet: Trace eyeblink conditioning decreases KCNN2 mRNA and protein levels in the hippocampus. A : KCNN2 protein levels (arbitrary units) as measured by Western analysis. B : KCNN2 mRNA levels normalized to beta 2-microglobulin as measured by qRT-PCR in the hippocampus

Article Snippet: Membranes were probed with one of two SK2 antibodies raised against two independent regions of the full-length SK2 protein (APC-028, 1:500; Alomone Labs, Jerusalem, Israel, aa 542–559; AV35094, Sigma, 1:500, aa 444–493) in 0.2% NFDM, 1% BSA TBS overnight at 4°C, followed by a 1-h incubation at 25°C with a horseradish peroxidase (HRP) conjugated secondary antibody (sc-2313, 1:5,000, Santa Cruz Biotechnology, Santa Cruz, CA) in 0.2% NFDM, 1% BSA TBS-T. Western blots were visualized by enhanced chemiluminescence (Immun-Star HRP, Bio-Rad, Hercules, CA) and developed on film (BioMax, Kodak, Carestream Health, Edison, NJ).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Pharmaceuticals

Article Title: The Inhibition of the Small-Conductance Ca2+-Activated Potassium Channels Decreases the Sinus Node Pacemaking during Beta-Adrenergic Activation

doi: 10.3390/ph15030313

Figure Lengend Snippet: ( A ) shows representative immunofluorescent images of rabbit sinus node cells with SK2 and HCN4 immunolabelling. ( B ) shows the relative expression of SK2 and HCN4. There were no significant changes between the SK2 and HCN4 fluorescence intensity. ( C ) shows fluorescence colocalization analysis. One data point represents one animal. Four cells were evaluated and averaged from each animal. Data are presented as mean ± SEM, applied statistical probe was unpaired Student’s t -test ( p ≤ 0.05).

Article Snippet: SK2 and HCN4 were labelled with anti-KCa2.2 (SK2) (Alomone, Jerusalem, Israel; 1:50) and anti-HCN4 (Alomone; 1:50) primary antibody overnight at 4 °C.

Techniques: Expressing, Fluorescence

Journal: Cell Calcium

Article Title: Functional distinctions in cytosolic calcium regulation between cells of the glomerular filtration barrier

doi: 10.1016/j.ceca.2010.06.005

Figure Lengend Snippet: Nephrin clustering affects the distribution of TRPC6 in HEK293 cells and potentially FFA induced changes in [Ca 2+ ] i . HEK293 were transiently transfected with TRPC6 and CD16-nephrin for 24 h. Cells were incubated with either secondary antibody alone (Ai–iii) or 1 mg/ml anti-CD16, then secondary antibody (Bi–iii) before fixing and immunostaining for TRPC6. Dapi nuclear staining is also shown in the merged images. Transfected HEKs, loaded with Fura-2AM were incubated in 1 mg/ml anti-CD16 for 10 min or vehicle, then incubated with 1 mg/ml mouse IgG for 10 min and finally stimulated with 200 μM FFA. Representative traces are shown for cells stimulated with FFA that were transfected with TRPC6 and pcDNA3 or TRPC6 and CD16-nephrin under control and experimental conditions (C). Data from C are summarised in (D) as area under the curve ( p

Article Snippet: 2.2 AntibodiesA rabbit polyclonal human anti-TRPC6 antibody (Alomone, Jerusalem, Israel) was used for immunofluorescence and Western blotting.

Techniques: Transfection, Incubation, Immunostaining, Staining

Journal: Cell Calcium

Article Title: Functional distinctions in cytosolic calcium regulation between cells of the glomerular filtration barrier

doi: 10.1016/j.ceca.2010.06.005

Figure Lengend Snippet: Expression and distribution of TRPC6 in ciGEnC and ciPod compared to a platelet positive control. Lysates from ciPod and cGEnC were Western blotted and probed using anti-TRPC6 and anti-actin antibodies. (A) Representative images of ciGEnC (Bi) and ciPod (Bii) were microinjected with pcDNA3TRPC6 and immunofluorescence was carried out using an anti-TRPC6 antibody. Cells were imaged using confocal microscopy. X–Y stacks of ciGEnC and ciPod in (B) are shown in (Ci) and (Cii) respectively.

Article Snippet: 2.2 AntibodiesA rabbit polyclonal human anti-TRPC6 antibody (Alomone, Jerusalem, Israel) was used for immunofluorescence and Western blotting.

Techniques: Expressing, Positive Control, Western Blot, Immunofluorescence, Confocal Microscopy