Journal: PLoS ONE

Article Title: Blockade of Kv1.3 Potassium Channels Inhibits Differentiation and Granzyme B Secretion of Human CD8+ T Effector Memory Lymphocytes

doi: 10.1371/journal.pone.0054267

Figure Lengend Snippet: A) Freshly isolated CD8+ T cells were pretreated with Kv channel blockers, ShK (10 nM), MgTx (30 nM) and ChTx (50 nM), for 3 hours, then stimulated with anti-CD3 alone or anti-CD3/CD28. After 4 days of culture, proliferation was measured by [3H] thymidine uptake. Data show the mean ± SD of three experiments. Significant differences are marked as follows: (*, p <0.05; **, p <0.01; ***, p <0.005). (B). Isolated CD8+ T cells were labeled with PKH26 stimulated with anti-CD3/CD28 for 24 h, and then transduced with a lentiviral vector encoding the dominant-negative Kv1.x or the GFP control alone. PKH26 fluorescence was analyzed by flow cytometry at baseline and 5 and 11 days later as shown. Quantification of proliferating cells was evaluated by gating on PKH26high PKH26dim and PKH26low among GFP+ cells. (C). Transduced CD8+ T cells were stained with anti-CD8, anti-CCR7 or anti-CD45RA mAbs seven days after transduction and analyzed for the percentages of naïve, T CM , T EMRA and T EM cells in gated GFP+ CD8+ cells. FACS plots shown are representative data from three separate experiments using cells from different donors. (D) The percentages of each CD8+ subset displaying GFP fluorescence are presented as mean ± SD of three experiments. Values that are significantly different from that of GFP control are indicated as follows: **, p <0.01; ***, p <0.005.

Article Snippet: Charybdotoxin (ChTx) and margatoxin (MgTx) were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Labeling, Transduction, Plasmid Preparation, Dominant Negative Mutation, Fluorescence, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Blockade of Kv1.3 Potassium Channels Inhibits Differentiation and Granzyme B Secretion of Human CD8+ T Effector Memory Lymphocytes

doi: 10.1371/journal.pone.0054267

Figure Lengend Snippet: Freshly isolated CD8+ T cells were pretreated with a Kv1.3 channel blocker, ShK at various concentrations (A) or with ShK (10 nM), MgTX (30 nM), ChTX (50 nM) and TRAM-34 (500 nM) (B) for 3 h, followed by stimulation with anti-CD3/CD28 or anti-CD3 alone. The levels of GrB were measured in cell supernatants by ELISA at 6 h (A) and indicated times (B and C). Data are mean of triplicate ± SD of one representative of three independent and reproducible experiments. Values that are significantly different from that of non-blocker vehicle treated control are indicated as follows: *, p <0.05; **, p <0.01.

Article Snippet: Charybdotoxin (ChTx) and margatoxin (MgTx) were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Blockade of Kv1.3 Potassium Channels Inhibits Differentiation and Granzyme B Secretion of Human CD8+ T Effector Memory Lymphocytes

doi: 10.1371/journal.pone.0054267

Figure Lengend Snippet: (A) Freshly isolated CD8+ T cells were stimulated with anti-CD3/CD28 or anti-CD3 for 24 hours. Cells were then stained with a CD107a-specific mAb, or an IgG1 isotype control (filled histogram) at the indicated times. (B). CD8+ T cells were pretreated with ShK (10 nM), MgTX (30 nM), ChTX (50 nM) and TRAM-34 (500 nM) for 3 h, followed by stimulation with anti-CD3/CD28 or anti-CD3 alone for 6 hours. Surface expression levels of CD107a were then analyzed by flow cytometry. FACS plots shown are representative data from three separate experiments.

Article Snippet: Charybdotoxin (ChTx) and margatoxin (MgTx) were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Staining, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Blockade of Kv1.3 Potassium Channels Inhibits Differentiation and Granzyme B Secretion of Human CD8+ T Effector Memory Lymphocytes

doi: 10.1371/journal.pone.0054267

Figure Lengend Snippet: Freshly isolated human CD8+ T cells were simulated with anti-CD3 or anti-CD3/CD28 in the presence or absence of MgTx. Cultured supernatants were collected at 3 days after stimulation. (A). Human neural cells cultured on poly-D-lysine pre-coated 96 well plates were pretreated with supernatants from non-activated CD8 T cells (Unstim.), anti-CD3 or anti-CD3/CD28-activated CD8+ T cells without MgTx (none), and with MgTx (MgTx). After 24 hours of treatment, CellQuanti-blue dye was added in each well for 30 minutes. Fluorescence was then detected using a plate reader. Cell viability was quantified by fluorescence intensity. (B). MgTx (30 nM) was added to culture media and incubated for 3 days. Human NPCs were treated with supernatants without MgTx (Ctrl), MgTx contained sups with vehicle treatment, with GrB alone (GrB) or MgTx containing sups plus GrB treatment (GrB/MgTx sups). Neurotoxicity was evaluated by cell viability quantified by fluorescence intensity. The fluorescence intensity in each group is plotted as percent relative to that in non-activated cells (Unstim.) or control cells (Ctrl). Data are mean of triplicate ± SD of one representative of three independent experiments. Values that are significantly different from that of vehicle treated control are indicated as *, p <0.05; **, p <0.01; ***, p <0.005.

Article Snippet: Charybdotoxin (ChTx) and margatoxin (MgTx) were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Cell Culture, Fluorescence, Incubation

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: TREK2 channel expression in human bladder cancer cells. (A) Messenger RNA (mRNA) of ion channels related to the TREK1 (Accession No; AF129399) and TREK2 (Accession No; AF279890) were amplified by reverse transcription-polymerase chain reaction (RT-PCR) analysis. TREK1 (355base pair [bp]) and TREK2 (291 bp) were detected. (B) Immunoblot showed presence of TREK2 channel protein in the human bladder cancer 253J cell line. TREK2 transfected CHO cells were used as a control. (C) Representative confocal microscopic analysis of TREK2 in bladder cancer cell line 253J. Cells were stained with an anti-TREK2 antibody and F-actin. Scale bar, 20 µm.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Staining

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: The physiologic properties of TREK2 at a single channel level in bladder cancer 253J cells. (A) TREK2 in CHO cells transfected with DNA encoding TREK2 and GFP and in 253J cells was measured in the excised inside-out patch configuration at the holding potential values shown on the left. The current trace was obtained in symmetrical 150 mM KCl solutions. The letters "c" and "o" represent the "closed" and "open" states of the channels, respectively. (B) The I-V relationships showed inward rectification, and each point is the mean of 4 experiments with standard error (S.E) represented by the error bars. (C~E) Current tracing showed arachidonic acid, intracellular pH, and mechanosensitivity of the native TREK2-like channel in 253J cells at -60 mV, +40 mV, and -40 mV. Negative pressure (-10 mmHg or -20 mmHg) was applied through the pipette. The panel below each of the figures shows the single channel trace on an expanded time scale.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Transfection, Transferring

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: The effect of TREK2 knockdown on the growth of 253J bladder cancer cells. (A and B) TREK2 mRNA and protein levels in 253J cell were determined after knockdown of TREK2 by siRNA through qRT-PCR and Western blot. Expression of TREK2 mRNA after transfection of TREK2 siRNA or negative control siRNA was normalized. TREK2 protein was examined by Western blot analysis after transfection of the negative control or TREK2 siRNA in 253J cells. GAPDH was used as a control. (C) The membrane potential was measured at current clamp (I=0) in a whole cell patch configuration. The 253J cells were treated with FITC-labeled negative control siRNA and FITC-labeled TREK2 siRNA for 72 hours. The data were represented as the mean±S.E. (t-test, p value <0.05). (D) The antiproliferative effect of TREK2 knockdown by siRNA in 253J cells. Cells were treated for three days with TREK2 siRNA or (-)control siRNA in 2% serum culture media. After treatment, proliferation was measured by XTT assay. Error bars represent the mean±S.E for 38 separate experiments. Asterisks indicate values that are different from the respective control (t-test, p<0.05). (E) Effect of TREK2 siRNA on 253J cell growth. Cells were captured 48 hours after transfection with TREK2 siRNA using a Nikon microscope at 10×10 magnification. Scale bar, 100 µm.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Negative Control, Labeling, XTT Assay, Microscopy

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: Cell cycle arrest at G0/G1 after TREK2 siRNA transfection in bladder cancer 253J cells. (A) The bar graph shows that TREK2 siRNA treated cells resulted in an increased percentage of cells in G0/G1 and a decreased percentage of cells in the S phase of the cell cycle (t-test p<0.05) (B) Representative Western blot showing changes in the levels of associated proteins in cell cycle arrest of human bladder cancer 253J cells. Knockdown of TREK2 decreased the expression of cyclin D1, cyclin D3, cdk2, cdk4, and cdk6 and increased protein levels of p21 and p53.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Transfection, Western Blot, Expressing

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Sound Rhythms Are Encoded by Postinhibitory Rebound Spiking in the Superior Paraolivary Nucleus

doi: 10.1523/JNEUROSCI.2450-11.2011

Figure Lengend Snippet: SPON neurons express HCN1 and HCN2 channels and have large Ih inwardly rectifying (IR) currents. A, HCN1 immunoreactivity in the superior olivary complex of an adult mouse. The low-magnification micrograph illustrates that the lateral superior olive expresses the strongest HCN1-like immunostaining, the SPON displays moderate levels of expression, and the MNTB shows the weakest immunoreactivity. At higher magnification, it is evident that both somata (arrowheads) and dendrites (arrow) of SPON neurons are immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. B) HCN2 is most strongly expressed in the MNTB, whereas expression in the LSO is weakest. At higher magnification, it is evident that SPON neurons are also immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. C, Current traces recorded from an SPON neuron induced by hyperpolarization from a holding current of −62 to −122 mV, in −10 mV voltage steps, under control conditions (top) and during pharmacological blockade of Ih with 20 μM ZD7288 (bottom). D, The size of the IR current was measured at steady state (i.e., ~1.35 s after induction) at each hyperpolarizing voltage (n = 42). The IR currents were significantly diminished by ZD7288 (n = 6) in the SPON neurons. ***p < 0.001, Student’s t test. E, Average activation time constants of the IR currents in SPON neurons were obtained by fitting a single-exponential function to the current traces.

Article Snippet: Sections were incubated in 2% normal donkey serum in blocking solution overnight at 4°C with one of the following primary antibodies: polyclonal rabbit α -HCN1 (1:250, lot number AN-10; Alomone Labs), which is directed against amino acid residues 6–24 of the intracellular N terminus of rat HCN1 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKB0","term_id":"29840774","term_text":"Q9JKB0"}} Q9JKB0 ); polyclonal rabbit α -HCN2 (1:400, lot number AN-08; Alomone Labs), directed against amino acids 147–161 of the intracellular N terminus of human HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 ); or monoclonal mouse α -HCN2 (1:400, clone N71/37; NeuroMab, UC Davis/NIH NeuroMab Facility) directed against amino acids 761–863 of the C terminus of rat HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKA9","term_id":"83303515","term_text":"Q9JKA9"}} Q9JKA9 ) used in combination with a mouse-on-mouse kit (Vector Laboratories).

Techniques: Immunostaining, Expressing, Activation Assay

Journal: PLoS ONE

Article Title: Up-Regulation of hERG K + Channels by B-RAF

doi: 10.1371/journal.pone.0087457

Figure Lengend Snippet: A. Representative original western blot showing hERG membrane protein abundance (anti-K v 11.1 antibody, Alamone Labs) analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 µM B-RAF inhibitor PLX-4720 (PLX-4720). B. Arithmetic means ± SEM (n = 7, arbitrary units) of normalized hERG membrane protein abundance analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 µM B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone. C. Representative original dot plots of hERG-FITC positive cells at the cell surface analysed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 µM B-RAF inhibitor PLX-4720 (PLX-4720); FL-1 Height: hERG-FITC fluorescence intensity. D. Arithmetic means ± SEM (n = 5, %) of normalized percentage of positive cells showing hERG expression at the cell surface analyzed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 µM B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone.

Article Snippet: After blocking with 5% non-fat dry milk in TBS 0.1% Tween20 for 1 hour at RT, the blots were incubated overnight at 4°C with rabbit anti-K v 11.1 (hERG, extracellular) antibody (diluted 1:200, Alamone Labs, Jerusalem, Israel).

Techniques: Western Blot, Flow Cytometry, Fluorescence, Expressing

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Enhanced large conductance K + channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

doi: 10.1152/ajpheart.00636.2013

Figure Lengend Snippet: Comparison of BK-α- and BK-β-subunit expression in cerebral vessel homogenates isolated from FHH and FHH.1BN rats. BK-α- and BK-β-subunits were probed on different blots. The same blots were then reprobed for β-actin. A: representative bands corresponding to the molecular weight of the BK-α-subunit (∼100 kDa), BK-β-subunit (∼25 kDa), and β-actin (∼42 kDa) in cerebral vessels obtained from FHH and FHH.1BN rats (n = 8 animals each; 3 sets of experiments). B: a comparison of the expression of α- and β-subunits. Numbers in parentheses indicate the number of samples studied.

Article Snippet: After transfer, the membrane was blocked with TBS-T buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween, and a 5% blocking powder (Bio-Rad) at 4°C for 1 h. The blot was probed with primary antibody against BK α- and β-subunit [1:500 and 1:200, respectively; polyclonal rabbit Anti-K Ca 1.1, to amino acids 1184–1200 and Anti-sloβ1 (KCNMB1); Alomone Labs, Jerusalem, Israel] overnight at 4°C.

Techniques: Expressing, Isolation, Molecular Weight