Journal: Frontiers in Pharmacology

Article Title: CyPPA, a Positive SK3/SK2 Modulator, Reduces Activity of Dopaminergic Neurons, Inhibits Dopamine Release, and Counteracts Hyperdopaminergic Behaviors Induced by Methylphenidate ¹

doi: 10.3389/fphar.2012.00011

Figure Lengend Snippet: CyPPA inhibits spontaneous and evoked release of dopamine form TH/SK3 positive cultured rat midbrain neurons . (A) Western blot detection of SK3 in rat midbrain cultures. Extracts of HEK293 cells transiently transfected with rSK1, rSK2, and rSK3 and adult rat brain membranes (RBM) as well as lysates from cultured midbrain neurons were separated on 4–15% SDS-PAGE and immunoblotted with anti-SK3 antibody. A band of approximately 70 kDa was detected in midbrain cultures, RBM lysates, and HEK293 cells transfected with rSK3. This band was absent in lanes loaded with HEK293 cells expressing rSK1 and rSK2. (B) Immunodetection of SK3 subunits in rat midbrain cultures. Cultured midbrain neurons (8 days in vitro ) were double-labeled for SK3 and tyrosine hydroxylase (TH). In TH-positive dopaminergic neurons, the SK3 subunit displayed a primarily somatic–dendritic localization whereas little immunoreactivity was associated with axons (arrow). Scale bar 20 μm. (C) Basal release and stimulated release with 25 mM KCl or inhibited release with 30 μM Cd 2+ and with 1 μM TTX. (D) Basal release and stimulated release with 3 μM haloperidol and inhibited release with 10 μM quinpirole. Blue bars represent inhibited release with increasing concentrations of CyPPA (* P < 0.05, ** P < 0.01). The data presented are representative of three independent experiments.

Article Snippet: Neurobasal media and B-27 were obtained from Invitrogen (Nærum, Denmark) and 3 H-DA was acquired from PerkinElmer Life and Analytical Sciences (Boston, MA, USA) The primary antibodies used were a rabbit polyclonal anti-SK3 antibody (1:100, APC-025) from Alomone Labs (Jerusalem, Israel) and a monoclonal mouse anti-tyrosine hydroxylase antibody (1:150, clone LNC1) from Millipore (Copenhagen, Denmark).

Techniques: Cell Culture, Western Blot, Transfection, SDS Page, Expressing, Immunodetection, In Vitro, Labeling

Journal: Biochemical and biophysical research communications

Article Title: Role of SK channel activation in determining the action potential configuration in freshly isolated human atrial myocytes from the SKArF study.

doi: 10.1016/j.bbrc.2019.03.074

Figure Lengend Snippet: Fig. 3. Presence of SK2 and SK3 channel subunits in human atrial myocytes. A. Confocal images (0.5-mm section) of a fixed and permeabilized cell labelled with anti-SK2 (green) and anti-SK3 (red). Labelling was observed at the cell periphery and superimposition of both images showed a significant degree of colocalization (yellow). B. Graph illustrates that an apparent similar level of labelling of SK2 and SK3 subunits in acutely isolated human atrial myocytes. C. Confocal images of SK2 (green) and SK3 (red) showing labelling at the cell edge. Superimposition of SK2 and SK3 channel subunit labelling demonstrates significant colocalization (yellow), which was quantified by using Mander's coefficient. Measured SK2 and SK3 labelling gave a Mander's coefficient of co-localization of SK3 overlapping SK2 by 0.94 ± 0.05 and SK2 overlapping SK3 by 093 ± 0.03. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cells were then incubated overnight at room temperature with mouse anti-SK2 antibody (Biorbyt: orb333955) and rabbit anti-SK3 antibody (Biorbyt: orb157735), added as a 1:200 dilution in blocking solution of composition (mM): phosphate buffer saline, bovine serum albumin (Sigma Aldrich, A9418) (1%), and foetal calf serum (Thermo Fisher Scientific, 10500-064) (2%) [20,21].

Techniques: Isolation

Journal: PLoS ONE

Article Title: CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

doi: 10.1371/journal.pone.0008959

Figure Lengend Snippet: A: The proportions of circulating CD4 T-cells that also expressed CD28 (CD28%) were reduced in many IPF patients. The horizontal line denotes the population means. B: In contrast to autologous CD4 + CD28 + cells, the CD4 + CD28 null T-cells of IPF patients more often express major histocompatibility antigen (MHC) Class II (DR), but less frequently express CD25. CD4 + CD28 null T-cells of IPF patients less frequently produce transcription factor FoxP3 (a putative marker of regulatory T-cells), but much more frequently produce cytotoxic mediators granzyme B (GB) and perforin (Perf). For each measure n = 24, and p values for all intergroup comparisons (CD4 + CD28 + vrs. CD4 + CD28 null cells) are <0.0001.

Article Snippet: Slides were incubated overnight at 4°C in blocking solution supplemented with rabbit polyclonal anti-human CD3 (ab5690, Abcam, Cambridge, MA), and mouse anti-human CD4 MAb (SK3, BD Biosciences).

Techniques: Marker

Journal: PLoS ONE

Article Title: CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

doi: 10.1371/journal.pone.0008959

Figure Lengend Snippet: Initial (left) data point in each series represents control unstimulated (basal) condition, while second (right) data point delineates productions of cells after stimulation with plate bound anti-CD3 antibody. These paired specimens (control and stimulated) are also connected by lines. CD4 + CD28 null cells from IPF patients (open circles with paired specimens connected by dashed lines) tend to elaborate greater amounts of pro-inflammatory and T H 1 cytokines (top two rows), whereas CD4 + CD28 + cells (open squares with paired specimens connected by solid lines) have an apparent T H 2 bias, with the exception of IL-4 production (bottom row) (n = 6 randomly-selected specimens in each measure).

Article Snippet: Slides were incubated overnight at 4°C in blocking solution supplemented with rabbit polyclonal anti-human CD3 (ab5690, Abcam, Cambridge, MA), and mouse anti-human CD4 MAb (SK3, BD Biosciences).

Techniques:

Journal: PLoS ONE

Article Title: CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

doi: 10.1371/journal.pone.0008959

Figure Lengend Snippet: Segregated autologous CD4 + CD28 + and CD4 + CD28 null cells from peripheral blood of IPF patients (n = 9) had similar proliferations (determined by BrdU incorporation) in 5-day control (unstimulated) cultures, as well as after stimulation with plate-bound anti-CD3 monoclonal antibody.

Article Snippet: Slides were incubated overnight at 4°C in blocking solution supplemented with rabbit polyclonal anti-human CD3 (ab5690, Abcam, Cambridge, MA), and mouse anti-human CD4 MAb (SK3, BD Biosciences).

Techniques: BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

doi: 10.1371/journal.pone.0008959

Figure Lengend Snippet: A.) Survival curves show cumulative freedom from major adverse events (lung transplantation or death) of IPF patients. Those subjects with the most extreme CD28 down-regulation, with CD28 expressed on <82% of their circulating CD4 T-cells (CD28% Low), had much worse outcomes than the cohort with greater proportions of CD4-Tcells that expressed CD28 (CD28% High). Numbers in parenthesis at the ends of survival curves denote remaining, unafflicted subjects that were censored at 12 months of observation. B.) Survival curves showing that cummulative freedom from major adverse events of IPF patients who have either significant CD28 downregulation (CD28% Low) or diffusing capacities for carbon monoxide, as percentages of predicted normal values (DLCO%p) <38, had worse outcomes than the IPF cohort who were both CD28% High and had more normal DLCO%p.

Article Snippet: Slides were incubated overnight at 4°C in blocking solution supplemented with rabbit polyclonal anti-human CD3 (ab5690, Abcam, Cambridge, MA), and mouse anti-human CD4 MAb (SK3, BD Biosciences).

Techniques: Transplantation Assay

Journal: PLoS ONE

Article Title: CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

doi: 10.1371/journal.pone.0008959

Figure Lengend Snippet: A.) and B.) Cells expressing CD4 (Green) and granzyme B (red), respectively, are widely distributed in IPF lung sections. C.) DAPI (blue) stains DNA within cell nuclei. D.) Co-localizations of CD4 and granzyme B (yellow) among nucleated cells are seen in the merged image. Nearly all the intrapulmonary CD4 + cells co-expressed granzyme B. Images E.) , F.) and G.) similarly depict CD4 (green), CD3 (red) and DAPI (blue). H.) Merged image shows most of the CD4 + cells also co-expressed CD3. Similar results were present in both IPF lungs. The majority of CD4 + cells in these IPF lung sections co-expressed both granzyme B and CD3 and are, thus, CD4 + CD28 null T-cells (see also ). All images are 60x.

Article Snippet: Slides were incubated overnight at 4°C in blocking solution supplemented with rabbit polyclonal anti-human CD3 (ab5690, Abcam, Cambridge, MA), and mouse anti-human CD4 MAb (SK3, BD Biosciences).

Techniques: Expressing

Journal: PLoS ONE

Article Title: CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

doi: 10.1371/journal.pone.0008959

Figure Lengend Snippet: Aliquots of fresh, live peripheral blood mononuclear cells (PBMNC) were stained with anti-CD4-allophycocyanin (APC), anti-CD28-fluorescein isothiocyanate (FITC), and phycoerythrin (PE)-conjugated antibodies against other cell epitopes. A.) Ten thousand (10,000) or more live cells were selected for further study, based their side scatter (SSC) and forward scatter (FSC) characteristics (G1). B.) The brightly staining CD4 cells among these also expressed CD3 (Cy-Chrome) and, thus, are T-cells . C.) These CD4 T-cells were further characterized based on their expression of CD28. The proportions of CD4 + CD28 + T-cells among the total CD4 + T-cell population (upper left and upper right quadrants) defines the CD28%. The respective proportions of CD4 + CD28 + and CD4 + CD28 null cells that co-expressed other cell determinants of interest (in this case MHC Class II [DR]) were quantitated. Numbers within the delineated region/quadrants denote the proportions of cells with these respective characteristics.

Article Snippet: Slides were incubated overnight at 4°C in blocking solution supplemented with rabbit polyclonal anti-human CD3 (ab5690, Abcam, Cambridge, MA), and mouse anti-human CD4 MAb (SK3, BD Biosciences).

Techniques: Staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Presence of CD8 + T Cells in the Ectocervical Mucosa Correlates with Genital Viral Shedding in HIV-Infected Women despite a Low Prevalence of HIV RNA-Expressing Cells in the Tissue

doi: 10.4049/jimmunol.1302826

Figure Lengend Snippet: Enumeration and in situ staining of immune cells in ectocervical tissue. (A) Distribution and median of the percentage of total cells in the ectocervical epithelium and submucosa of tissue samples from the three study groups. A nonparametric, two-tailed Mann–Whitney U test was used to compare the HIV+FSW group versus the HIV−FSW group and the HIV+FSW group versus the HIV−LR group. (B) Immunofluorescent images of ectocervical tissue sections from an HIV+FSW subject. The image on the right for each pair is a magnified view of the region indicated in the box in the image to the left. The majority of CD8+ cells (red) were also CD3+ (green) and vice versa (left panels); the majority of CD3+ cells (red) were not CD4+ cells (green) (right panels). Scale bar, 100 μm. (C) Bright-field images of ectocervical tissue stained with hematoxylin (blue) for visualization of cell nuclei and stained for CD8+ cells (brown; upper panels) and CD3+ cells (brown; lower panels). Scale bar, 200 μm.

Article Snippet: Thereafter, CD8 + cells were detected with an Alexa Fluor 647–conjugated monoclonal mouse anti-human CD8 Ab (RPA-T8; BD Pharmingen), and CD4 + cells were detected with a monoclonal mouse anti-human CD4 Ab (clone SK3; BD Biosciences), followed by the addition of an Alexa Fluor 488–conjugated donkey anti-mouse IgG Ab (Molecular Probes).

Techniques: In Situ, Staining, Two Tailed Test, MANN-WHITNEY