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: 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: Journal of Cerebral Blood Flow & Metabolism

Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for neuroinflammation associated with ischemia/reperfusion stroke

doi: 10.1177/0271678X15611434

Figure Lengend Snippet: KCa3.1 and Kv1.3 are expressed on microglia/macrophages in human infarcts. (a) KCa3.1 staining in a 2–3-week-old infarct. KCa3.1 expression is localized to macrophages/microglia (M) and vascular endothelial (E) cells. (b) Fluorescent staining for a microglia/macrophage marker (MAC387) and KCa3.1. (c) Kv1.3 staining in a 14-day old-infarct. (d) Fluorescent staining for a microglia/macrophage marker (MAC387) and Kv1.3. All images are from 5-μm thick paraffin sections.

Article Snippet: KCa3.1 was stained for with a rabbit polyclonal anti-KCa3.1 antibody (1:3000, AV35098, Sigma-Aldrich) and Kv1.3 with a mouse monoclonal anti-human Kv1.3 antibody (1:100; 1D8, AbD Serotec).

Techniques: Staining, Expressing, Marker

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: The potassium channel KCa3.1 constitutes a pharmacological target for neuroinflammation associated with ischemia/reperfusion stroke

doi: 10.1177/0271678X15611434

Figure Lengend Snippet: K+-channel expression in acutely isolated microglia. (a) Kv1.3 current density increases in microglia from the infarct area after MCAO (28.8 ± 2.0 pA/pF, n = 19) and microglia isolated from the hippocampus following intracerebroventricular LPS injection (22.9 ± 16.6 pA/pF, n = 13) compared to microglia from wild-type control brains (5.0 ± 3.9 pA/pF, n = 16) or microglia from the contralateral side after MCAO (5.7 ± 4.4 pA/pF, n = 18). (b) Example current traces showing Kv1.3's characteristic use-dependence and sensitivity to the Kv1.3 blockers PAP-1 and ShK-L5. (c) Microglia from the contralateral (50.2 ± 35.4 pS/pF, n = 18) and ipsilateral side after MCAO (71.6 ± 34.9 pS/pF, n = 21), as well as microglia isolated from the hippocampus following intracerebroventricular LPS injection (84.0 ± 42.4 pS/pF, n = 13) show higher KCa3.1 current densities than microglia from wild-type control brains (29.7 ± 15.2 pS/pF, n = 16). (d) Example KCa3.1 current traces elicited by a ramp protocol showing the current's sensitive to 1 µM of the KCa3.1-selective blocker TRAM-34. (e) Microglia from both the contralateral side (7.8 ± 5.8 pA/pF, n = 18) and the infarct area (15.1 ± 10.2 pA/pF, n = 21) after MCAO show increased Kir current densities compared to those from wild-type (2.4 ± 2.4 pA/pF, n = 16) or LPS-injected brains (1.9 ± 2.9 pA/pF, n = 13). (f) Representative current traces showing a large Kir current, which was observable in some MCAO microglia, but not in microglia isolated from the hippocampus following intracerebroventricular LPS injection. Data are presented as mean ± S.D. Statistical significance was determined by Student's t-test.

Article Snippet: KCa3.1 was stained for with a rabbit polyclonal anti-KCa3.1 antibody (1:3000, AV35098, Sigma-Aldrich) and Kv1.3 with a mouse monoclonal anti-human Kv1.3 antibody (1:100; 1D8, AbD Serotec).

Techniques: Expressing, Isolation, Injection