Journal: Cell calcium

Article Title: Differential modulation of SK channel subtypes by phosphorylation.

doi: 10.1016/j.ceca.2020.102346

Figure Lengend Snippet: Fig. 1. The apparent Ca2+ sensitivity of SK1 and IK channel subtypes is more decreased than that of the SK3 and SK2 subtypes in immortal ized endothelial cells. Concentration-dependent activation by Ca2+ of the SK channel subtypes heterologously expressed in HEK293 cells (A) and immortalized endothelial cells (B). (C) EC50 values for the activation by Ca2+, recorded from SK channel subtypes expressed in HEK293 cells (HEK) and immortalized endothelial cells (ET). All data are shown as mean ± S.E.M (n = 6-11, **P < 0.01, ***P < 0.001, Two-way ANOVA followed by Tukey’s post hoc tests).

Article Snippet: The proteins were transferred to PVDF membranes and incubated overnight at 4 °C with primary GFP antibody (1:2000; Invitrogen, Lot: 2,015,993), SK1 antibody (1:1000; RayBiotech, Lot: 907,002,018), SK2 antibody (1:200; Alomone Labs, Lot: APC028AN2325), SK3 antibody (1:200; Alomone Labs, Lot: APC025AN1125), IK antibody (1:1000; Santa Cruz, Lot: I2319), mitochondria marker Cytochrome C antibody (1:1000; Novus Biologicals, Lot: AB0115109A-2), ER marker GRP78/HSPA5 (1:4000; Novus Biologicals, Lot: H-1) antibody or plasma membrane marker sodium potassium ATPase alpha 1 (1:4000; Novus Biologicals, Lot: B-5) antibody.

Techniques: Concentration Assay, Activation Assay

Journal: British Journal of Pharmacology

Article Title: NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

doi: 10.1111/bph.12049

Figure Lengend Snippet: Primers used for the RT-PCR experiments

Article Snippet: Cells were washed two times with PBS, blocked and permeabilized in PBS containing 10% normal donkey serum and 0.1% Triton X-100 for 30 min. Once again, cells were washed with PBS and incubated with primary antibodies: anti-K Ca 2.1 (KCNN1, SK1); anti-K Ca 2.2 (KCNN2, SK2); anti-K Ca 2.3 (KCNN3, SK3) or anti-K Ca 3.1 (KCNN4, IK, SK4); 1:100; Alomone Labs (Jerusalem, Israel) at 37°C for 1 h. Next, cells were washed two times with PBS and labelled with secondary antibodies (Cy3-conjugated anti-rabbit IgG, at 1:100, PBS/3% normal donkey serum/0.01% Triton X-100; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h in the dark.

Techniques:

Journal: British Journal of Pharmacology

Article Title: NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

doi: 10.1111/bph.12049

Figure Lengend Snippet: RT-PCR detection of mRNA messages for SK3 channels in DSM whole tissue and freshly isolated single cells. mRNA expression for the SK3 channel was detected in DSM whole tissue and freshly isolated DSM single cells, whereas no mRNA expression was observed for SK1, SK2 and IK (SK4) channels in DSM single cells. Rat brain was used as a positive control. Negative control experiments carried out with omission of RT-enzyme (−RT) in reaction mixtures showed no products. Illustrated gel images are representations of at least four independent RT-PCR experiments based on RNA extracted from four rats.

Article Snippet: Cells were washed two times with PBS, blocked and permeabilized in PBS containing 10% normal donkey serum and 0.1% Triton X-100 for 30 min. Once again, cells were washed with PBS and incubated with primary antibodies: anti-K Ca 2.1 (KCNN1, SK1); anti-K Ca 2.2 (KCNN2, SK2); anti-K Ca 2.3 (KCNN3, SK3) or anti-K Ca 3.1 (KCNN4, IK, SK4); 1:100; Alomone Labs (Jerusalem, Israel) at 37°C for 1 h. Next, cells were washed two times with PBS and labelled with secondary antibodies (Cy3-conjugated anti-rabbit IgG, at 1:100, PBS/3% normal donkey serum/0.01% Triton X-100; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h in the dark.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Positive Control, Negative Control

Journal:

Article Title: Mechanism of Differential Cardiovascular Response to Propofol in Dahl Salt-Sensitive, Brown Norway, and Chromosome 13-Substituted Consomic Rat Strains: Role of Large Conductance Ca 2+ and Voltage-Activated Potassium Channels

doi: 10.1124/jpet.109.154104

Figure Lengend Snippet: Expression of BK α and β1 subunits in small mesenteric arteries of SS and BN rats. a, representative images of BK α subunit immunofluorescence staining in SS and BN rats. The expression of α and β1 subunits was assessed by confocal microscopy using selective polyclonal antibodies and the fluorescence-tagged secondary antibody. b, quantification by densitometry demonstrated that expression of α subunit was significantly greater in SS compared with BN rats. Although β1 subunit expression also varied between SS and BN rats, the difference was not significant.

Article Snippet: Thereafter, the vessels were incubated for 1 h at 37°C with the rabbit polyclonal primary antibodies for BK channel α subunit (anti-KCa 2+ 1.1/KCNMA1; Alomone Labs, Jerusalem, Israel; 1:50 dilution in PBS) and β 1 subunit (anti-slob1/KCNMB1; Alomone Labs; 1:100 dilution in PBS).

Techniques: Expressing, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence