Journal: Journal for Immunotherapy of Cancer

Article Title: PD1 blockade enhances K + channel activity, Ca 2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer

doi: 10.1136/jitc-2020-000844

Figure Lengend Snippet: Study design scheme and experimental protocol. (A) Samples were collected from patients with head and neck squamous cell carcinoma in an ongoing clinical trial (top; NCT02641093) or from patients undergoing standard of care treatment (bottom) using an institutional review board-approved sample collection protocol. A treatment-naive (TN) blood sample was received from each patient before any treatment. Pembrolizumab patients then received a single 200 mg dose of pembrolizumab infusion. Approximately 1–3 weeks after the infusion, a post-treatment (PT) blood sample was obtained. Patients both in the pembrolizumab and control groups underwent surgical resection of the tumor. Anywhere from 4 to 11 weeks post-resection (PR), patients then had a blood sample drawn prior to adjuvant treatment (chemotherapy/radiation) (pembrolizumab patients: PR-αPD1; control patients: PR-CTR). (B) CD8 + PBTs were isolated from whole blood of pembrolizumab and control patients. Experiments were conducted on both resting (r-PBTs) and activated (a-PBTs) CD8 + PBTs. In r-PBTs, electrophysiology was used to determine KCa3.1 and Kv1.3 activities. Ca 2+ fluxes were also measured. Flow cytometry was used to determine T cell phenotype and ion channel expression. In a-PBTs, in addition to K + channel activity and Ca 2+ fluxes, chemotaxis was measured. (C) Surgically resected solid tumors from pembrolizumab and control patients were dissociated and the single-cell suspension was used to determine T cell phenotype and ion channel expression. CD8 + tumor infiltrating lymphocytes (TILs) were then isolated from the remaining single-cell suspension. KCa3.1 and Kv1.3 activities and Ca 2+ fluxes were measured in the CD8 + TILs. αPD1, anti-programmed death 1 antibody; PBTs, peripheral blood T cells.

Article Snippet: PBTs or TILs were stained for surface KCa3.1 (6C1/ATTO-488; Alomone Labs) and Kv1.3 (polyclonal; Alomone Labs) primary antibody, followed by a secondary anti-guinea pig (Dy350 goat anti-guinea pig IgG; ThermoFisher), Orai1 (polyclonal; Alomone Labs) primary followed by a secondary anti-rabbit (Alexa Fluor 594 Goat Anti-Rabbit IgG; Jackson ImmunoResearch Laboratories).

Techniques: Isolation, Flow Cytometry, Expressing, Activity Assay, Chemotaxis Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: PD1 blockade enhances K + channel activity, Ca 2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer

doi: 10.1136/jitc-2020-000844

Figure Lengend Snippet: Pembrolizumab increases Kv1.3 activity and Ca 2+ fluxes in TILs from non-responders and responders. (A) Representative KCa3.1 and Kv1.3 current traces recorded in TILs from control (CTR) and pembrolizumab (αPD1) patients. Currents were normalized for the maximum current recorded at +50 mV. (B) KCa3.1 (left) and Kv1.3 (right) activities reported as conductance (G) from individual TILs from CTR (n=36 and 35 cells, respectively, from 11 patients), non-responder (NR) (n=46 and 45 cells from 10 patients), and responder (R) (n=19 cells from 6 patients) patients. On average we recorded 4–5 cells/patient. Data compared via Kruskal-Wallis one-way ANOVA on ranks (p=0.105 left; p=0.003 right). (C) Representative single-cell intracellular Ca 2+ recordings in TILs from CTR and αPD1 patients. The time of TG application is indicated by an arrow. (D) ΔCa 2+ in CTR (n=117 cells from 12 patients; average of 10 cells/patient), NR (n=257 cells from 10 patients; average of 26 cells/patient), and R (n=94 cells from 6 patients; average of 16 cells/patient) patients. Data compared via Kruskal-Wallis one-way ANOVA on ranks (p=0.008). (B, D) Data are represented as box plots: the line indicates the median; the lower box is the 25th percentile; the upper box is the 75th percentile; and the whiskers represent the 10th and 90th percentiles. αPD1, anti-programmed death 1 antibody; ANOVA, analysis of variance; TG, thapsigargin; TILs, tumor infiltrating lymphocytes.

Article Snippet: PBTs or TILs were stained for surface KCa3.1 (6C1/ATTO-488; Alomone Labs) and Kv1.3 (polyclonal; Alomone Labs) primary antibody, followed by a secondary anti-guinea pig (Dy350 goat anti-guinea pig IgG; ThermoFisher), Orai1 (polyclonal; Alomone Labs) primary followed by a secondary anti-rabbit (Alexa Fluor 594 Goat Anti-Rabbit IgG; Jackson ImmunoResearch Laboratories).

Techniques: Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: PD1 blockade enhances K + channel activity, Ca 2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer

doi: 10.1136/jitc-2020-000844

Figure Lengend Snippet: Pembrolizumab increases KCa3.1 activity short term and Kv1.3 activity long term in responder PBTs. (A) Representative KCa3.1 and Kv1.3 currents recorded in activated PBTs from pembrolizumab patients at treatment-naive (TN; never received any treatment) and post-treatment (PT; after pembrolizumab) time points. Currents were normalized to PT for the maximum current recorded at +50 mV. (B) KCa3.1 (left) and Kv1.3 (right) activities reported as conductance (G) from TN (n=160 cells from 32 patients) and PT (n=118 cells from 22 patients) activated PBTs. (C) KCa3.1 (left) and Kv1.3 (right) activities (G) from individual post-resection (PR) activated PBTs from control (PR-CTR; PBTs obtained after tumor resection from patients who did not receive pembrolizumab treatment) (n=34 and 33 cells, respectively, from 7 patients) and pembrolizumab (PR-αPD1; PBTs obtained after tumor resection from patients who were treated with pembrolizumab) (n=79 and 78 cells, respectively, from 15 patients) patients. (D) KCa3.1 (left) and Kv1.3 (right) activities (G) from PT activated PBTs from non-responder (NR) (n=68 cells from 12 patients) and responder (R) (n=50 cells from 10 patients) patients and from individual PR activated PBTs from NR (n=44 and 43 cells, respectively, from 8 patients) and R (n=35 cells from 7 patients) patients. (E) Kv1.3 activity (G) from individual PR resting PBTs (r-PBTs) from NR (n=40 cells from 8 patients) and R (n=35 cells from 7 patients) patients. (B–E) Data are represented as box plots: the line indicates the median; the lower box is the 25th percentile; the upper box is the 75th percentile; and the whiskers represent the 10th and 90th percentiles. Data compared using Mann-Whitney rank-sum test. On average we recorded 4–5 cells/patient. αPD1, anti-programmed death 1 antibody; PBTs, peripheral blood T cells.

Article Snippet: PBTs or TILs were stained for surface KCa3.1 (6C1/ATTO-488; Alomone Labs) and Kv1.3 (polyclonal; Alomone Labs) primary antibody, followed by a secondary anti-guinea pig (Dy350 goat anti-guinea pig IgG; ThermoFisher), Orai1 (polyclonal; Alomone Labs) primary followed by a secondary anti-rabbit (Alexa Fluor 594 Goat Anti-Rabbit IgG; Jackson ImmunoResearch Laboratories).

Techniques: Activity Assay, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: PD1 blockade enhances K + channel activity, Ca 2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer

doi: 10.1136/jitc-2020-000844

Figure Lengend Snippet: Pembrolizumab increases memory subset percentage in responder PBTs and increases Temra and Teff KCa3.1 expression post-resection. (A) Cells were gated based on the following: live cells, lymphocytes, CD8 + cells and then CD45RO/CD45RA. Percentage of resting PBTs composed of naive (Ra + ) or memory (RO + ) phenotypes post-treatment (PT; left) (NR patients n=11; R patients n=9) and post-resection (PR; right) (NR patients n=10; R patients n=7) was determined in non-responder (NR) and responder (R) patients. (B) Cells were gated based on the following: live cells, lymphocytes, CD8 + cells and then memory subtypes using CD45RA/CD127. Percentage of PT resting PBTs (left) composed of Tnaive, Temra, Tm, and Teff subsets (NR patients n=10; R patients n=7) was determined in NR and R patients. Percentage of PR resting PBTs (right) composed of Tnaive, Temra, Tm, and Teff subsets (NR patients n=9; R patients n=5) was determined in NR and R patients. (C, D) Cells were gated based on the following: live cells, lymphocytes, CD8 + cells, memory subtypes using CD45RA/CD127 and then ion channel expression (left: KCa3.1; right: Kv1.3). (C) Normalized KCa3.1 MFI (left) (NR patients n=10; R patients n=6) and normalized Kv1.3 MFI (right) (NR patients n=10; R patients n=7) in PT resting PBTs within the Tnaive, Temra, Tm, and Teff subsets were determined in NR and R patients. KCa3.1 data compared using Student’s t-test (Temra, Teff) or Mann-Whitney rank-sum test (Tnaive, Tm). Kv1.3 data compared using Student’s t-test (Tnaive, Temra, Tm) or Mann-Whitney rank-sum test (Teff). (D) Normalized KCa3.1 MFI (left) (NR patients n=9; R patients n=4) and normalized Kv1.3 MFI (right) (NR patients n=9; R patients n=5) in PR resting PBTs within the Tnaive, Temra, Tm, and Teff subsets were determined in NR and R patients. Data compared using Student’s t-test. (A–D) All data are represented as mean±SEM and compared via Student’s t-test unless otherwise indicated. MFI, mean fluorescence intensity; PBTs, peripheral blood T cells; Teff, effector T cells; Temra, effector memory reexpressing CD45RA T cells; Tm, memory T cells; Tnaive, naive T cells.

Article Snippet: PBTs or TILs were stained for surface KCa3.1 (6C1/ATTO-488; Alomone Labs) and Kv1.3 (polyclonal; Alomone Labs) primary antibody, followed by a secondary anti-guinea pig (Dy350 goat anti-guinea pig IgG; ThermoFisher), Orai1 (polyclonal; Alomone Labs) primary followed by a secondary anti-rabbit (Alexa Fluor 594 Goat Anti-Rabbit IgG; Jackson ImmunoResearch Laboratories).

Techniques: Expressing, MANN-WHITNEY, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: PD1 blockade enhances K + channel activity, Ca 2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer

doi: 10.1136/jitc-2020-000844

Figure Lengend Snippet: Proposed model of cytotoxic T cell function in non-responder and responder patients. Summary of functional aspects in CD8 + T cells of non-responder and responder patients post-treatment and post-resection. In non-responders, post-treatment activated PBTs have increased KCa3.1 activity. Additionally, these activated PBTs are able to chemotax; however, in the presence of adenosine, the chemotaxis is inhibited. In the tumor, the non-responders have low T cell infiltration; however, the T cells that do infiltrate have increased Kv1.3 activity and Ca 2+ fluxing ability. After tumor removal (post-resection), the activated PBTs have increased Kv1.3 activity, but these cells are not able to chemotax. On the other hand, in responders, post-treatment activated PBTs have increased KCa3.1 and Kv1.3 activities and Ca 2+ fluxing ability. Additionally, these activated PBTs are able to chemotax even in the presence of adenosine. Furthermore, in the tumors of responders, there is higher T cell infiltration and these cells, like the non-responders, have increased Kv1.3 activity and Ca 2+ fluxing ability. Moreover, after tumor removal, resting PBTs of responders already have increased Kv1.3 activity and in the activated PBTs the Kv1.3 activity is also increased. Additionally, these cells are able to chemotax, although their chemotaxis is inhibited by adenosine. PBTs, peripheral blood T cells. Created with BioRender.com.

Article Snippet: PBTs or TILs were stained for surface KCa3.1 (6C1/ATTO-488; Alomone Labs) and Kv1.3 (polyclonal; Alomone Labs) primary antibody, followed by a secondary anti-guinea pig (Dy350 goat anti-guinea pig IgG; ThermoFisher), Orai1 (polyclonal; Alomone Labs) primary followed by a secondary anti-rabbit (Alexa Fluor 594 Goat Anti-Rabbit IgG; Jackson ImmunoResearch Laboratories).

Techniques: Cell Function Assay, Functional Assay, Activity Assay, Chemotaxis Assay

Journal: Frontiers in Immunology

Article Title: Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19

doi: 10.3389/fimmu.2023.1143350

Figure Lengend Snippet: Changes in ion channel expression with disease progression and dexamethasone treatment. Fold change in mRNA abundance of Kv1.3 ( KCNA3 , A ), KCa3.1 ( KCNN4 , B ), Orai ( ORAI1 , C ) and Stim1 ( STIM1 , D ) in PBMCs from healthy donors (n=4), and patients with mild COVID-19 (n=4), severe COVID-19 (Severe, n=3), and severe COVID-19 + dexamethasone (Dexa, n=4) was determined by RT-qPCR. Data are normalized to healthy donor PBMCs. Each sample was run in triplicate. 18S rRNA was used as the housekeeping gene. Bars represent means ± SD, and each symbol represents an individual patient. Data were analyzed by one-way analysis of variance (ANOVA) (P < 0.001 in A , P = 0.4270 in B , P=0.0003 in C, and P=0.0414 in D ), and post hoc testing was performed by the Holm-Sidak method.

Article Snippet: Cells were stained for surface guinea pig anti- Kv1.3 (Alomone Labs) and Orai1 (ATTO-633, Alomone Labs) primary antibodies overnight at 4°C in the dark.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19

doi: 10.3389/fimmu.2023.1143350

Figure Lengend Snippet: Effect of dexamethasone on Kv1.3 channel abundance. (A) Kv1.3 ( KCNA3 ), Orai1 ( ORAI1 ), and Stim1 ( STIM1 ) mRNA expression in PBMCs from healthy donors (n=4) treated with 0.1 μM and 1μM dexamethasone or vehicle for 48 h was determined by RT-qPCR. Data were normalized to vehicle-treated control. Significance was determined by one-way analysis of variance (ANOVA) for KCNA3 (P < 0.001), ORAI1 (P=0.005), and by ANOVA on ranks for STIM1 (p=0.630). Post-hoc testing was performed by the Holm-Sidak method. (B) Kv1.3 and Orai1 abundance in immune cell subsets from healthy donor PBMCs treated with 1 μM dexamethasone for 48 h. (Left) Representative flow cytometry histograms showing Kv1.3 (top) and Orai1 (bottom) abundance in the presence or absence of dexamethasone treatment in CD3 + T cell subsets. (Right) Mean fluorescence intensity (MFI) of Kv1.3 and Orai1 in immune cell subsets from three healthy donors treated in vitro with 1 μM dexamethasone or vehicle for 48 h. Significance determined by paired t-test. means ± SD, and each symbol represents an individual donor. .

Article Snippet: Cells were stained for surface guinea pig anti- Kv1.3 (Alomone Labs) and Orai1 (ATTO-633, Alomone Labs) primary antibodies overnight at 4°C in the dark.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Fluorescence, In Vitro

Journal: Frontiers in Immunology

Article Title: Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19

doi: 10.3389/fimmu.2023.1143350

Figure Lengend Snippet: Effect of dexamethasone on Kv1.3 channel function, Ca 2+ signaling and cytokine production. (A) Inhibition of Kv1.3 currents in CD8 + T cells treated with 1 μM dexamethasone (Dexa) for 24 and 48 h. Representative Kv1.3 currents are shown on the left, and percentage inhibition of Kv1.3 currents by dexamethasone in n=4 donors is shown on the right. (B) Representative Ca 2+ response (shown as a ratio of Indo-1 fluorescence at 400 and 480 nm) recorded in activated healthy donor CD8 + T cells treated with either 1μM dexamethasone, 10 nM ShK-Dap22, or vehicle for 48 h are shown on the left. Cells were loaded with Indo-1 ratiometric dye, and the fluorescence was recorded by flow cytometry. Indo-1 loaded cells were first exposed to thapsigargin (arrow) in 0 mM Ca 2+ , followed by 2 mM Ca 2+ , which yields a rapid influx of Ca 2+ (see Materials and Methods). Data are representative of independent experiments performed in CD8 + T cells isolated from n=3 healthy donors. The average fold change in peak Ca 2+ levels in n = 3 healthy donors are shown on the right. Significance was determined by one-way analysis of variance (ANOVA, p=0.0309), and post hoc testing was performed by Tukey’s test. (C) IFN-γ release determined by ELISA in the supernatant of activated CD8 + T cells from n=4 healthy donors and n=3 severe COVID-19 patients. Significance was determined by unpaired t test (D) Percent inhibition of IFN-γ secretion as compared to vehicle treated cells after treatment of activated healthy donor (n=4) CD8 + T cells with either 0.1 μM and 1μM dexamethasone, 10 nM and 100 nM ShK-Dap22, or vehicle for 48 h. Significance was determined by one-way analysis of variance (ANOVA, p=0.9390). (E) Percent inhibition of IFN-γ secretion compared to vehicle treated cells after treatment of activated severe COVID-19 patient (n=3) CD8 + T cells with either 1μM dexamethasone, 10 nM ShK-Dap22, or vehicle for 48 h. Significance was determined by paired-t test. Activation of CD8 + T cells in panels (B–E) was done for 48 h with plate bound anti-CD3 and anti-CD28 antibodies. Bars represent means ± SD, and each symbol represents an individual.

Article Snippet: Cells were stained for surface guinea pig anti- Kv1.3 (Alomone Labs) and Orai1 (ATTO-633, Alomone Labs) primary antibodies overnight at 4°C in the dark.

Techniques: Inhibition, Fluorescence, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: International Journal of Nephrology

Article Title: Overexpression of Delayed Rectifier K + Channels Promotes In situ Proliferation of Leukocytes in Rat Kidneys with Advanced Chronic Renal Failure

doi: 10.1155/2012/581581

Figure Lengend Snippet: Kv1.3 and cell cycle marker expression in sham operated (sham) and advanced CRF rat kidneys. (A) Kv1.3 expression. (a) KCNA3 mRNA abundance in the renal cortex of sham operated (sham) and advanced CRF (CRF) rat kidneys. (b) and (c) Immunohistochemistry using antibody for Kv1.3 (brown) in sham operated and advanced CRF rat kidneys. High-power views of cortical interstitium. Magnification, ×60. (B) Cell cycle marker expression. The mRNA abundance of cyclin-dependent kinase 4 (Cdk4) (left) and p21 (right) in the renal cortex of sham operated and advanced CRF rat kidneys. # P < 0.05 versus sham operated rats. Values are means ± SEM ( n = 6). Differences were analyzed by ANOVA followed by Dunnett's or Student's t -test.

Article Snippet: Primary antibodies were as follows: rabbit anti-Ki-67 (1 : 100; Lab Vision Co., Fremont, CA, USA), anti-Kv1.3 (1 : 100; Alomone Labs Ltd., Jerusalem, Israel), and mouse anticollagen type III (1 : 100; Abnova, Taipei City, Taiwan).

Techniques: Marker, Expressing, Immunohistochemistry

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) Purified CD8+ T cells were stimulated with anti-CD3/CD28 for 3 days. Naïve and stimulated cells were then immunostained for Kv1.3 in combination with CD8 and subsequently viewed by immunofluorescence microscopy. Cellular nuclei were counterstained with DNA dye DAPI (blue). Kv1.3 detected by AF 594 fluorescence is shown in red, while CD8 detected by AF 488 fluorescence is shown in green. Colocalization is indicated by a yellow and/or orange color in the overlay panels. (B) An isotype-matched antibody was used as a negative control. Original magnification, ×100. Image is representative of three different donors. (C) Summary of percentages of activated CD8+ T cells expressing Kv1.3. In brief, 4 view fields/microscopic section were evaluated for Kv1.3+ CD8 cells stimulated with anti-CD3/CD28 or anti-CD3 alone for 3 days. The percentages of Kv1.3+ cells are based on the number of CD8+ T cells counted. Data are mean ± SD from one representative of three independent and reproducible experiments. Values that are significantly different from that of non-stimulated control are indicated as **, p <0.01.

Article Snippet: Thereafter, cells were incubated with rabbit anti-human Kv1.3 (Alomone Labs, Jerusalem, Israel), mouse anti-human GrB (Caltag Laboratories, San Francisco, CA) or mouse anti-human CD8 (PharMingen) antibodies for 30 min at room temperature.

Techniques: Purification, Immunofluorescence, Microscopy, Fluorescence, Negative Control, Expressing

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: Thereafter, cells were incubated with rabbit anti-human Kv1.3 (Alomone Labs, Jerusalem, Israel), mouse anti-human GrB (Caltag Laboratories, San Francisco, CA) or mouse anti-human CD8 (PharMingen) antibodies for 30 min at room temperature.

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: (A) Naïve, T CM , T EMRA and T EM CD8 subpopulations were sorted from CD8+ gated cell population based on surface markers of CCR7 and CD45RA. Sorted individual subsets within the respective gates shown were transduced with a DN-Kv1.x and GFP control. After 7 days of transfection, the percentages of each subset in gated GFP+ CD8+ cells were analyzed by flow cytometry. Gate for expression of GFP was established using untransduced controls. (B) FACS profiles are representative of three separate experiments using cells from different donors. The percentage of cells in each quadrant is indicated. (C) The percentages of CD8 subsets displaying GFP fluorescence from each single transfected subpopulation are presented as mean ± SD of three experiments. Values that are significantly different from that of GFP control are indicated as follows: *, p <0.05, **, p <0.01; ***, p <0.005. (D) Representative FACS profiles of phenotypical changes of transduced T CM and T EM subsets 21 days after transfection. (E). FACS-sorted CCR7- (T EM /T EMRA ) were labeled with PKH26 day (2×10–6 M), followed by stimulation with anti-CD3/CD28 for 24 h and then transduced with a lentiviral vector encoding the dominant-negative Kv1.x and GFP control alone at an MOI of ∼8. PKH26 fluorescence was analyzed by flow cytometry at days 0, 5 and 11. FACS plots shown are representative data from two experiments.

Article Snippet: Thereafter, cells were incubated with rabbit anti-human Kv1.3 (Alomone Labs, Jerusalem, Israel), mouse anti-human GrB (Caltag Laboratories, San Francisco, CA) or mouse anti-human CD8 (PharMingen) antibodies for 30 min at room temperature.

Techniques: Transduction, Transfection, Flow Cytometry, Expressing, Fluorescence, Labeling, Plasmid Preparation, Dominant Negative Mutation

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: Thereafter, cells were incubated with rabbit anti-human Kv1.3 (Alomone Labs, Jerusalem, Israel), mouse anti-human GrB (Caltag Laboratories, San Francisco, CA) or mouse anti-human CD8 (PharMingen) antibodies for 30 min at room temperature.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid) matrix nanoparticles on human monocytes

doi: 10.2147/IJN.S106540

Figure Lengend Snippet: Demonstration of voltage-gated potassium channels in primary cells and the MM6 cells. Notes: Western blot of voltage-gated potassium channels ( A ) Kv1.3 and ( B ) Kv7.1 in the leukemic monocyte cell line MM6 and primary monocytes (M0). Primary antibodies used were rabbit anti-Kv1.3 (3 µg/mL; APC-002; Alomone Labs) or rabbit anti-Kv7.1 (4 µg/mL; AB5932; Millipore, Billerica, MA, USA) and a goat anti-rabbit IgG coupled to alkaline phosphatase (50 ng/mL; Abcam). Bound antibody was stained by the NBT/BCIP technique (Sigma-Aldrich Co.). Abbreviations: IgG, immunoglobulin G; NBT/BCIP, nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indoylphosphate p -toluidine salt.

Article Snippet: The used antibodies were anti-Kv1.3 (APC-002) (Alomone Labs, Jerusalem, Israel), anti-Kv7.1 (AB5932) (Merck Millipore), unspecific rabbit IgG (ab27478) (Abcam), and goat anti-rabbit IgG (A11012) (Thermo Fisher Scientific).

Techniques: Western Blot, Staining

Journal: International Journal of Nanomedicine

Article Title: Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid) matrix nanoparticles on human monocytes

doi: 10.2147/IJN.S106540

Figure Lengend Snippet: Validation of voltage-gated potassium channels. Notes: Fluorescent staining of the voltage-gated potassium channel proteins ( A and B ) Kv1.3 and ( C and D ) Kv7.1 in ( A , C , and E ) primary monocytes or ( B , D , and F ) the MM6 cell line with an ( E and F ) unspecific IgG control. Cells were stained with either rabbit anti-Kv1.3 (3 µg/mL; APC-002; Alomone Labs) or rabbit anti-Kv7.1 (4 µg/mL; AB5932; Millipore) or unspecific rabbit IgG (4 µg/mL; ab27478; Abcam) and goat anti-rabbit IgG (10 µg/mL; A11012; Thermo Fisher Scientific) coupled to Alexa Fluor 594. Abbreviation: IgG, immunoglobulin G.

Article Snippet: The used antibodies were anti-Kv1.3 (APC-002) (Alomone Labs, Jerusalem, Israel), anti-Kv7.1 (AB5932) (Merck Millipore), unspecific rabbit IgG (ab27478) (Abcam), and goat anti-rabbit IgG (A11012) (Thermo Fisher Scientific).

Techniques: Staining

Journal: International Journal of Nanomedicine

Article Title: Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid) matrix nanoparticles on human monocytes

doi: 10.2147/IJN.S106540

Figure Lengend Snippet: Kv currents of Xenopus laevis oocytes in two-electrode voltage clamp experiments. Notes: Effects of PLGA nanoparticles or SPIONs on Xenopus laevis oocytes expressing ( A ) Kv1.3 and ( B ) Kv7.1. The oocytes were incubated with 500 ng/mL PLGA nanoparticles or SPIONs for 4 hours. The black curve (n=24/22) indicates the untreated control oocytes, and the currents are normalized to this curve. The blue curve (n=21/28) indicates the treatment with PLGA nanoparticles, the red curve (n=21/17) indicates treatment with SPIONs, the green curve (n=12/14) represents control oocytes without RNA injection and nanoparticle treatment as an example for all uninjected oocytes with and without nanoparticle treatment. Abbreviations: PLGA, poly(lactic-co-glycolic acid); SPIONs, starch-coated superparamagnetic iron oxide nanoparticles.

Article Snippet: The used antibodies were anti-Kv1.3 (APC-002) (Alomone Labs, Jerusalem, Israel), anti-Kv7.1 (AB5932) (Merck Millipore), unspecific rabbit IgG (ab27478) (Abcam), and goat anti-rabbit IgG (A11012) (Thermo Fisher Scientific).

Techniques: Expressing, Incubation, Injection

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Expression of human Kv1.3 in Tetrahymena thermophila . A. Expression construct design. KCNA3 , the gene encoding human Kv1.3, was modified with a C-terminal FLAG/10Xhis tag and placed under the control of the MTT5 and MTT1 promoter and terminator, respectively. The entire expression cassette was cloned as a NotI fragment into an rDNA vector, pTRAS1. The relative positions of chromosome breakage sites (CBS) and ribosomal genes (17s, 5.8s, and 26s) are shown. B. Single cell isolates maintain expression of recombinant Kv1.3. Anti-Kv1.3 Western analysis of single cells isolated from pooled Tetrahymena transformants and tested for their ability to express Kv1.3. Eight of nine single cell isolates expressed Kv1.3 at similar levels to the original pool (T1) with one clone (#117) expressing higher-levels of Kv1.3. A lysate from wild-type cells (WT) was included as a negative control. C. Tetrahymena -expressed Kv1.3 is phosphorylated. Purified Kv1.3 was incubated in the absence (−) and presence (+) of calf-intestinal alkaline phosphatase (CIP) and subsequently detected by anti-Kv1.3 Western analysis as described above. D. Comparison of Kv1.3 expression levels in Tetrahymena and CHO cells. Cell lysates generated from 25,000 Kv1.3 expressing Tetrahymena (Tth) or CHO cells were resolved by SDS-PAGE. Kv1.3 was detected by Western analysis using an anti-Kv1.3 antibody and an anti-guinea pig HRP conjugated antibody. E. Tetrahymena -expressed Kv1.3 binds both Agitoxin-2 (AgTX-2) and ShK. Mock-induced wild-type cells (WT) and Kv1.3-expressing Tetrahymena cells were fixed and labeled with either 10 nM Agitoxin-2-TAMRA (AgTX-2-TAMRA) or ShK-TAMRA and visualized by fluorescence confocal microscopy. Inset shows a close-image of a single Tetrahymena cell. White arrows highlight the Tetrahymena plasma (surface) membrane. F. Binding of ShK to Tetrahymena Kv1.3 is specific. Fixed Tetrahymena cells expressing Kv1.3 were incubated with 10 nM ShK-TAMRA in the presence of saturating (10X) amounts of Margatoxin (MgTx) or Iberiotoxin (IbTx) and examined by fluorescence confocal microscopy.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Expressing, Construct, Modification, Clone Assay, Plasmid Preparation, Recombinant, Western Blot, Isolation, Negative Control, Purification, Incubation, Generated, SDS Page, Labeling, Fluorescence, Confocal Microscopy, Binding Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Purification of recombinant Kv1.3. a. SDS-PAGE analysis of purified and reconstituted Kv1.3. Kv1.3 was purified as described in Materials, resolved by SDS-PAGE before and after reconstitution into liposomes and stained with SimplyBlue™ SafeStain™. B. Ligand Binding Analysis. Kv1.3-containing liposomes were incubated with FAM-ShK (3 nM) in the presence or absence of a 50-fold excess of either MgTx or IbTx. Top Panel is a representative experiment showing total binding expressed as Anisotropy measured by fluorescence polarization. Bottom Panel represents specific binding to FAM-ShK. Kd was estimated as 11.5 nM +/− 3.4 nM based on specific binding curves generated in three separate experiments c. Fluorescence microscopy analysis of Kv1.3 magnetic beads. Magnetic beads containing tethered Kv1.3 reconstituted into a lipid bilayer consisting of rhodamine-labeled PE and non-labeled PC were examined by fluorescence microscopy. Beads were examined under a rhodamine filter to detect labeled PE incorporation (Left Panel) and with a FITC-filter following labelling with an anti-Kv1.3 antibody that recognizes an epitope on the first extracellular loop and anti-guinea pig conjugated FITC (Middle Panel). Rhodamine and FITC- images were merged (Right Panel) to confirm co-localization of Kv1.3 and the lipid bilayer (yellow fluorescence). D. Schematic illustration of Kv1.3 magnetic beads. Shown is the magnetic bead surface; the lipid bilayer consisting of PC (yellow lipids) and Rhodamine-labeled PE (red lipids); the six transmembrane domains of the Kv1.3 monomer (S1-S6); the C-terminal engineered FLAG (orange Triangle) and 10Xhis (Green Box) tags; a star indicates the position of the epitope on the first extracellular loop that is recognized by the Kv1.3 antibody utilized in c and e. e. Kv1.3 magnetic beads preferentially precipitate an antibody that recognizes an extracellular epitope. Kv1.3 magnetic beads or control beads were incubated with antibodies (6.7 nM) that recognize either internal (FLAG and 10Xhis) or external (Kv1.3) epitopes. Beads were washed and bound IgG eluted directly in SDS-PAGE loading buffer. IgG was detected by Western analysis using either anti-mouse-HRP (anti-FLAG and -His) or anti-guinea pig-HRP (anti-Kv1.3).

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Purification, Recombinant, SDS Page, Staining, Ligand Binding Assay, Incubation, Binding Assay, Fluorescence, Generated, Microscopy, Magnetic Beads, Labeling, Western Blot

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Summary of anti-Kv1.3 antibody identification.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Enzyme-linked Immunosorbent Assay, Functional Assay, Magnetic Beads

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Identification of anti-Kv1.3 antibodies. A. Chicken anti-Kv1.3 antibodies. ScFv-Fc screening was carried out by ELISA using three-fold serial dilutions of antibody against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . An isotype control (IC) for generated antibodies was also included. Shown are results for antibodies that inhibit Kv1.3 activity. Note the relative lack of reactivity of clone ch_p1E6 against Kv1.3 and clone p1F8 reactivity against the irrelevant proteoliposome control. B. Llama anti-Kv1.3 antibodies. ScFv-Fc screening was carried out using ten-fold serial dilutions of antibody on mesoscale against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . IC1, isotype control for generated antibodies; IC2, isotype control for anti-Kv1.3 polyclonal antibody. Data is shown for 6 of 19 specific Kv1.3 binding scFv-Fc antibodies.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Enzyme-linked Immunosorbent Assay, Generated, Activity Assay, Binding Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Identification of anti-Kv1.3 antibodies that functionally inhibit Kv1.3 channel activity. Purified scFv-Fc anti-Kv1.3 antibodies from either chickens or llamas were tested at a concentration of 400nM via electrophysiology for their ability to block current from human Kv1.3 expressed in L929 fibroblast cells. Shown are representative traces for each of the antibodies that blocked Kv1.3 current. Black lines represent control currents, red lines represent currents following addition of antibody. Inhibiting anti-Kv1.3 antibody clones derived from chickens are shown in a, the functional llama anti-Kv1.3 antibody is shown in b. An example of an antibody that was tested and shown not to modulate Kv1.3 activity is shown in c. d. Time-dependent development of current inhibition by monoclonal antibodies targeting Kv1.3. (Left Panel) Time-current plots showing current inhibition of three (3) individual cells expressing hKv1.3 channels by the monoclonal antibody ScFv-Fc L1A3. Antibodies were added after current stabilization at 0 second. Currents were elicited by pulsing to +40 mV for 200 ms from a holding potential of −80 mV every 30 seconds. (Right Panel) Means ± SD plot of the current inhibition of three individual cells in the left panel. Similar time-dependent profiles were observed for each of the blocking antibodies.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Activity Assay, Purification, Concentration Assay, Blocking Assay, Clone Assay, Derivative Assay, Functional Assay, Inhibition, Expressing

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Summary of anti-Kv1.3 antibody functional activity.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Functional Assay, Activity Assay, Blocking Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Anti-Kv1.3 antibody potency analysis. A. Dose response curves. Ten-fold serial dilutions of chicken antibody p1E6 (red traces) and llama antibody L1A3 (green traces) were used to generate dose-response curves. Black lines represent control currents. B. IC 50 Determinations. IC 50 s for p1E6 (red curve) and L1A3 (green curve) were determined by fitting the calculated percentage of current block to a Hill equation. IC 50 s for p1E6 and L1A3 were estimated as 6 and 109 nM, respectively c. Analysis of p1E6 and L1A3 selectivity. ScFv-Fc clones p1E6 and L1A3 were tested for their ability to block the activity of related Kv1.x family members (Kv1.1, Kv1.2, Kv1.5), hERG and Nav1.5 at a concentration of 1 μM. Shown are representative traces from each experiment. Black lines, control current; Red lines, currents following addition of antibody.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Blocking Assay, Clone Assay, Activity Assay, Concentration Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: L1A3 and p1E6 selectivity profile.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques:

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Kv1.3 epitope binning analysis. A. Binning analysis heat map. Shown are antibodies colored by bin (1–5) and by functional data. Antibodies that block Kv1.3 current are highlighted in blue (Ephys only), those that additionally bind Jurkat cells are highlighted in red (Ephys + Jurkat) and those that do neither are highlighted in grey. Relative competition activity of each antibody is color coded and indicates strong competition (red boxes), intermediate/weak competition (yellow boxes) or no competition (green boxes). Dark red boxes indicate competition from the same antibody pair. B. Binning network plot. Antibodies are colored to identify those that inhibit channel activity (blue), additionally bind Jurkat cells (red) or do neither (grey). Note antibody L1A3 was not tested for its ability to bind Jurkat cells, however, for simplicity was denoted as inhibiting ion channel only (blue) in a & b.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Functional Assay, Blocking Assay, Activity Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Kv1.3 ortholog sequence alignment analysis.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Sequencing

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Expression of human Kv1.3 in Tetrahymena thermophila . A. Expression construct design. KCNA3 , the gene encoding human Kv1.3, was modified with a C-terminal FLAG/10Xhis tag and placed under the control of the MTT5 and MTT1 promoter and terminator, respectively. The entire expression cassette was cloned as a NotI fragment into an rDNA vector, pTRAS1. The relative positions of chromosome breakage sites (CBS) and ribosomal genes (17s, 5.8s, and 26s) are shown. B. Single cell isolates maintain expression of recombinant Kv1.3. Anti-Kv1.3 Western analysis of single cells isolated from pooled Tetrahymena transformants and tested for their ability to express Kv1.3. Eight of nine single cell isolates expressed Kv1.3 at similar levels to the original pool (T1) with one clone (#117) expressing higher-levels of Kv1.3. A lysate from wild-type cells (WT) was included as a negative control. C. Tetrahymena -expressed Kv1.3 is phosphorylated. Purified Kv1.3 was incubated in the absence (−) and presence (+) of calf-intestinal alkaline phosphatase (CIP) and subsequently detected by anti-Kv1.3 Western analysis as described above. D. Comparison of Kv1.3 expression levels in Tetrahymena and CHO cells. Cell lysates generated from 25,000 Kv1.3 expressing Tetrahymena (Tth) or CHO cells were resolved by SDS-PAGE. Kv1.3 was detected by Western analysis using an anti-Kv1.3 antibody and an anti-guinea pig HRP conjugated antibody. E. Tetrahymena -expressed Kv1.3 binds both Agitoxin-2 (AgTX-2) and ShK. Mock-induced wild-type cells (WT) and Kv1.3-expressing Tetrahymena cells were fixed and labeled with either 10 nM Agitoxin-2-TAMRA (AgTX-2-TAMRA) or ShK-TAMRA and visualized by fluorescence confocal microscopy. Inset shows a close-image of a single Tetrahymena cell. White arrows highlight the Tetrahymena plasma (surface) membrane. F. Binding of ShK to Tetrahymena Kv1.3 is specific. Fixed Tetrahymena cells expressing Kv1.3 were incubated with 10 nM ShK-TAMRA in the presence of saturating (10X) amounts of Margatoxin (MgTx) or Iberiotoxin (IbTx) and examined by fluorescence confocal microscopy.

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques: Expressing, Construct, Modification, Clone Assay, Plasmid Preparation, Recombinant, Western Blot, Isolation, Negative Control, Purification, Incubation, Generated, SDS Page, Labeling, Fluorescence, Confocal Microscopy, Binding Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Purification of recombinant Kv1.3. a. SDS-PAGE analysis of purified and reconstituted Kv1.3. Kv1.3 was purified as described in Materials, resolved by SDS-PAGE before and after reconstitution into liposomes and stained with SimplyBlue™ SafeStain™. B. Ligand Binding Analysis. Kv1.3-containing liposomes were incubated with FAM-ShK (3 nM) in the presence or absence of a 50-fold excess of either MgTx or IbTx. Top Panel is a representative experiment showing total binding expressed as Anisotropy measured by fluorescence polarization. Bottom Panel represents specific binding to FAM-ShK. Kd was estimated as 11.5 nM +/− 3.4 nM based on specific binding curves generated in three separate experiments c. Fluorescence microscopy analysis of Kv1.3 magnetic beads. Magnetic beads containing tethered Kv1.3 reconstituted into a lipid bilayer consisting of rhodamine-labeled PE and non-labeled PC were examined by fluorescence microscopy. Beads were examined under a rhodamine filter to detect labeled PE incorporation (Left Panel) and with a FITC-filter following labelling with an anti-Kv1.3 antibody that recognizes an epitope on the first extracellular loop and anti-guinea pig conjugated FITC (Middle Panel). Rhodamine and FITC- images were merged (Right Panel) to confirm co-localization of Kv1.3 and the lipid bilayer (yellow fluorescence). D. Schematic illustration of Kv1.3 magnetic beads. Shown is the magnetic bead surface; the lipid bilayer consisting of PC (yellow lipids) and Rhodamine-labeled PE (red lipids); the six transmembrane domains of the Kv1.3 monomer (S1-S6); the C-terminal engineered FLAG (orange Triangle) and 10Xhis (Green Box) tags; a star indicates the position of the epitope on the first extracellular loop that is recognized by the Kv1.3 antibody utilized in c and e. e. Kv1.3 magnetic beads preferentially precipitate an antibody that recognizes an extracellular epitope. Kv1.3 magnetic beads or control beads were incubated with antibodies (6.7 nM) that recognize either internal (FLAG and 10Xhis) or external (Kv1.3) epitopes. Beads were washed and bound IgG eluted directly in SDS-PAGE loading buffer. IgG was detected by Western analysis using either anti-mouse-HRP (anti-FLAG and -His) or anti-guinea pig-HRP (anti-Kv1.3).

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques: Purification, Recombinant, SDS Page, Staining, Ligand Binding Assay, Incubation, Binding Assay, Fluorescence, Generated, Microscopy, Magnetic Beads, Labeling, Western Blot

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Summary of anti-Kv1.3 antibody identification.

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques: Enzyme-linked Immunosorbent Assay, Functional Assay, Magnetic Beads

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Identification of anti-Kv1.3 antibodies. A. Chicken anti-Kv1.3 antibodies. ScFv-Fc screening was carried out by ELISA using three-fold serial dilutions of antibody against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . An isotype control (IC) for generated antibodies was also included. Shown are results for antibodies that inhibit Kv1.3 activity. Note the relative lack of reactivity of clone ch_p1E6 against Kv1.3 and clone p1F8 reactivity against the irrelevant proteoliposome control. B. Llama anti-Kv1.3 antibodies. ScFv-Fc screening was carried out using ten-fold serial dilutions of antibody on mesoscale against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . IC1, isotype control for generated antibodies; IC2, isotype control for anti-Kv1.3 polyclonal antibody. Data is shown for 6 of 19 specific Kv1.3 binding scFv-Fc antibodies.

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques: Enzyme-linked Immunosorbent Assay, Generated, Activity Assay, Binding Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Identification of anti-Kv1.3 antibodies that functionally inhibit Kv1.3 channel activity. Purified scFv-Fc anti-Kv1.3 antibodies from either chickens or llamas were tested at a concentration of 400nM via electrophysiology for their ability to block current from human Kv1.3 expressed in L929 fibroblast cells. Shown are representative traces for each of the antibodies that blocked Kv1.3 current. Black lines represent control currents, red lines represent currents following addition of antibody. Inhibiting anti-Kv1.3 antibody clones derived from chickens are shown in a, the functional llama anti-Kv1.3 antibody is shown in b. An example of an antibody that was tested and shown not to modulate Kv1.3 activity is shown in c. d. Time-dependent development of current inhibition by monoclonal antibodies targeting Kv1.3. (Left Panel) Time-current plots showing current inhibition of three (3) individual cells expressing hKv1.3 channels by the monoclonal antibody ScFv-Fc L1A3. Antibodies were added after current stabilization at 0 second. Currents were elicited by pulsing to +40 mV for 200 ms from a holding potential of −80 mV every 30 seconds. (Right Panel) Means ± SD plot of the current inhibition of three individual cells in the left panel. Similar time-dependent profiles were observed for each of the blocking antibodies.

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques: Activity Assay, Purification, Concentration Assay, Blocking Assay, Clone Assay, Derivative Assay, Functional Assay, Inhibition, Expressing

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Summary of anti-Kv1.3 antibody functional activity.

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques: Functional Assay, Activity Assay, Blocking Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Anti-Kv1.3 antibody potency analysis. A. Dose response curves. Ten-fold serial dilutions of chicken antibody p1E6 (red traces) and llama antibody L1A3 (green traces) were used to generate dose-response curves. Black lines represent control currents. B. IC 50 Determinations. IC 50 s for p1E6 (red curve) and L1A3 (green curve) were determined by fitting the calculated percentage of current block to a Hill equation. IC 50 s for p1E6 and L1A3 were estimated as 6 and 109 nM, respectively c. Analysis of p1E6 and L1A3 selectivity. ScFv-Fc clones p1E6 and L1A3 were tested for their ability to block the activity of related Kv1.x family members (Kv1.1, Kv1.2, Kv1.5), hERG and Nav1.5 at a concentration of 1 μM. Shown are representative traces from each experiment. Black lines, control current; Red lines, currents following addition of antibody.

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques: Blocking Assay, Clone Assay, Activity Assay, Concentration Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: L1A3 and p1E6 selectivity profile.

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques:

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Kv1.3 epitope binning analysis. A. Binning analysis heat map. Shown are antibodies colored by bin (1–5) and by functional data. Antibodies that block Kv1.3 current are highlighted in blue (Ephys only), those that additionally bind Jurkat cells are highlighted in red (Ephys + Jurkat) and those that do neither are highlighted in grey. Relative competition activity of each antibody is color coded and indicates strong competition (red boxes), intermediate/weak competition (yellow boxes) or no competition (green boxes). Dark red boxes indicate competition from the same antibody pair. B. Binning network plot. Antibodies are colored to identify those that inhibit channel activity (blue), additionally bind Jurkat cells (red) or do neither (grey). Note antibody L1A3 was not tested for its ability to bind Jurkat cells, however, for simplicity was denoted as inhibiting ion channel only (blue) in a & b.

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques: Functional Assay, Blocking Assay, Activity Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Kv1.3 ortholog sequence alignment analysis.

Article Snippet: Anti-FLAG antibody was from Thermo Fisher Scientific (#MA1-91878), anti-C-terminal His antibody was from Life Technologies (#R93025), anti-Kv1.3 antibody was from Alomone (#AGP-005).

Techniques: Sequencing