rabbit anti kv1 1  (Alomone Labs)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization

doi: 10.1073/pnas.1608763114

Figure Lengend Snippet: Kv1.1 channels determine spike width. ( A ) Example recordings from one neuron before and after 20 nM DTx-K application. This neurotoxin had no effect on somatic spikes but led to broadening of presynaptic action potentials. ( B ) Presynaptic spike widths

Article Snippet: Membranes were immunoblotted with the respective antibodies: rabbit anti-Kv1.1 (APC-161, Alomone Labs; 1:800), anti-Kv1.2 (APC-010, 1:1,500), anti-Kv1.3 (APC-101, 1:1,000), anti-Kv1.4 (APC-167, 1:500), anti-Kv1.6 (APC-003, 1:2,000), and anti-SNAP25 (Abcam ab5666; 1:2,000) at 4 °C overnight.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization

doi: 10.1073/pnas.1608763114

Figure Lengend Snippet: Blockade or deletion of Kv1.1 does not prevent analog modulation of presynaptic spike width. ( A ) Trace analysis from one wild-type cell showing bidirectional changes in presynaptic spike width by subthreshold somatic current injections before evoking

Article Snippet: Membranes were immunoblotted with the respective antibodies: rabbit anti-Kv1.1 (APC-161, Alomone Labs; 1:800), anti-Kv1.2 (APC-010, 1:1,500), anti-Kv1.3 (APC-101, 1:1,000), anti-Kv1.4 (APC-167, 1:500), anti-Kv1.6 (APC-003, 1:2,000), and anti-SNAP25 (Abcam ab5666; 1:2,000) at 4 °C overnight.

Techniques:

Journal: The Journal of Physiology

Article Title: Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons

doi: 10.1113/jphysiol.2001.018333

Figure Lengend Snippet: Kv1.1, Kv1.2 and Kv1.6 are detected in Western blots of nodose ganglia and brain protein Western blots of channel expression in nodose ganglia ( A , B and C ), brain lysates ( A and B ) and brain crude membrane fraction ( C ) probed with monoclonal anti-Kv1.1 ( A ), monoclonal anti-Kv1.2 ( B ) and polyclonal anti-Kv1.6 ( C ) (50 μg protein per lane). Immunoreactive bands were visualized with ECL-Plus (Amersham Pharmacia Biotech). Molecular weight markers (kDa) are indicated on the left.

Article Snippet: Two Kv1.1 antibodies were used: a mouse monoclonal antibody (Upstate Biotechnology) raised against a C-terminal peptide (residues 458-476) of rat Kv1.1 and a rabbit polyclonal Kv1.1 (Alomone Labs) raised against a fusion protein with sequence corresponding to residues 416-495.

Techniques: Western Blot, Expressing, Molecular Weight

Journal: BMC Biology

Article Title: The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K+ channel Kv1.1 through protein kinase C delta

doi: 10.1186/s12915-020-00878-1

Figure Lengend Snippet: ARTD10 inhibition reduces the proportion of the inactivating Kv1.1 current and enhances spontaneous excitation in hippocampal neurons. a Left, whole cell currents of mouse hippocampal neurons in the presence of tetrodotoxin with and without an inhibitor of ARTD10 (OUL35). Right, bar graphs representing I steady-state / I peak ( I s / I p ) and peak current amplitudes. b Resting membrane potential (RMP) and spontaneous spikes/s from control and OUL35 treated hippocampal neurons. * p

Article Snippet: For immunoblots, proteins were transferred to PVDF membranes (Roche, Mannheim, Germany), and the membrane was blocked for 1 h at RT in 5% non-fat milk in TBS-T (137 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween-20), and probed overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Kv1.1 (Alomone Labs, # APC-161 and APC-009), rabbit polyclonal anti-PKCδ (Santa Cruz, # sc-937), rabbit polyclonal anti-phospho-PKCδ (Tyr311) (Cell Signaling, # 2055), rat monoclonal anti-ARTD10 (Merck, #5H11), or mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, # T7451).

Techniques: Inhibition

Journal: BMC Biology

Article Title: The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K+ channel Kv1.1 through protein kinase C delta

doi: 10.1186/s12915-020-00878-1

Figure Lengend Snippet: Scheme illustrating the interplay of PKA, PKCδ, and ARTD10 in the regulation of Kv1.1. The regulation of a phosphatase by PKCδ is hypothetical. Current traces above the schemes of the Kv1.1 channels illustrate the typical inactivation pattern of phosphorylated vs. un-phosphorylated Kv1.1α. I/F, IBMX/forskolin; PMA, phorbol-myristate-acetate

Article Snippet: For immunoblots, proteins were transferred to PVDF membranes (Roche, Mannheim, Germany), and the membrane was blocked for 1 h at RT in 5% non-fat milk in TBS-T (137 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween-20), and probed overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Kv1.1 (Alomone Labs, # APC-161 and APC-009), rabbit polyclonal anti-PKCδ (Santa Cruz, # sc-937), rabbit polyclonal anti-phospho-PKCδ (Tyr311) (Cell Signaling, # 2055), rat monoclonal anti-ARTD10 (Merck, #5H11), or mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, # T7451).

Techniques:

Journal: BMC Biology

Article Title: The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K+ channel Kv1.1 through protein kinase C delta

doi: 10.1186/s12915-020-00878-1

Figure Lengend Snippet: ARTD10 inhibition enhances excitability of hippocampal neurons via Kv1.1. a Left, representative current clamp recordings of APs elicited by step current pulses in control neurons and neurons treated with OUL35. Right, bar graphs represent the rheobase and the latency to the first spike. For cells with a RMP more positive than − 60 mV, the membrane potential was adjusted to ~ − 60 mV. b The number of spikes elicited by step current pulses were counted and for stimuli from 10 to 30 pA they were fitted with a linear function. Right, bar graphs summarize the AP amplitude from neurons with and without OUL35 treatment. c Left, bar graphs representing the rheobase. Right, summary of spikes/s with and without the Kv1 inhibitor α-dendrotoxin (DTX) and from neurons with and without OUL35 treatment. * p

Article Snippet: For immunoblots, proteins were transferred to PVDF membranes (Roche, Mannheim, Germany), and the membrane was blocked for 1 h at RT in 5% non-fat milk in TBS-T (137 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween-20), and probed overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Kv1.1 (Alomone Labs, # APC-161 and APC-009), rabbit polyclonal anti-PKCδ (Santa Cruz, # sc-937), rabbit polyclonal anti-phospho-PKCδ (Tyr311) (Cell Signaling, # 2055), rat monoclonal anti-ARTD10 (Merck, #5H11), or mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, # T7451).

Techniques: Inhibition

Journal: BMC Biology

Article Title: The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K+ channel Kv1.1 through protein kinase C delta

doi: 10.1186/s12915-020-00878-1

Figure Lengend Snippet: Inactivation of Kv1.1 is regulated by Kvβ and phosphorylation at S446. a Representative recordings, I steady-state / I peak ( I s / I p ), and peak current amplitudes of Kv1.1 with or without co-expression of Kvβ1.1 in HeLa cells. b Representative recordings and I s / I p before and after the application of either IBMX/forskolin (I/F) or of the phorbol ester PMA. c Representative recordings, I s / I p , and peak current amplitudes of the phosphorylation-deficient mutant Kv1.1 S446A co-expressed with Kvβ1.1. d Representative recordings, I s / I p , and peak current amplitudes of Kv1.1 after 1.5 h pre-incubation with a phosphatase inhibitor cocktail. * p

Article Snippet: For immunoblots, proteins were transferred to PVDF membranes (Roche, Mannheim, Germany), and the membrane was blocked for 1 h at RT in 5% non-fat milk in TBS-T (137 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween-20), and probed overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Kv1.1 (Alomone Labs, # APC-161 and APC-009), rabbit polyclonal anti-PKCδ (Santa Cruz, # sc-937), rabbit polyclonal anti-phospho-PKCδ (Tyr311) (Cell Signaling, # 2055), rat monoclonal anti-ARTD10 (Merck, #5H11), or mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, # T7451).

Techniques: Expressing, Mutagenesis, Incubation

Journal: BMC Biology

Article Title: The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K+ channel Kv1.1 through protein kinase C delta

doi: 10.1186/s12915-020-00878-1

Figure Lengend Snippet: ARTD10 leads to phosphorylation of Kv1.1 at S446. a , b Left, Representative current traces of wild type ( a ) or the phosphorylation-deficient mutant ( b ) of Kv1.1 overexpressed in three different HeLa cell lines. Right, bar graphs representing I steady-state / I peak ( I s / I p ) and peak current amplitudes. * p

Article Snippet: For immunoblots, proteins were transferred to PVDF membranes (Roche, Mannheim, Germany), and the membrane was blocked for 1 h at RT in 5% non-fat milk in TBS-T (137 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween-20), and probed overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Kv1.1 (Alomone Labs, # APC-161 and APC-009), rabbit polyclonal anti-PKCδ (Santa Cruz, # sc-937), rabbit polyclonal anti-phospho-PKCδ (Tyr311) (Cell Signaling, # 2055), rat monoclonal anti-ARTD10 (Merck, #5H11), or mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, # T7451).

Techniques: Mutagenesis

Journal: BMC Biology

Article Title: The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K+ channel Kv1.1 through protein kinase C delta

doi: 10.1186/s12915-020-00878-1

Figure Lengend Snippet: Kv1.1 is regulated by PKCδ in HeLa cells. a , b Wild type Kv1.1 or mutant Kv1.1-S446A were co-expressed with Kvβ1 and either the catalytic domain (CAT) or a dominant negative mutant (DN) of PKCδ. Bar graphs represent I steady-state / I peak ( I s / I p ) and peak current amplitudes

Article Snippet: For immunoblots, proteins were transferred to PVDF membranes (Roche, Mannheim, Germany), and the membrane was blocked for 1 h at RT in 5% non-fat milk in TBS-T (137 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween-20), and probed overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Kv1.1 (Alomone Labs, # APC-161 and APC-009), rabbit polyclonal anti-PKCδ (Santa Cruz, # sc-937), rabbit polyclonal anti-phospho-PKCδ (Tyr311) (Cell Signaling, # 2055), rat monoclonal anti-ARTD10 (Merck, #5H11), or mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, # T7451).

Techniques: Mutagenesis, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Compartment-Specific Neurexin Nanodomains Orchestrate Tripartite Synapse Assembly

doi: 10.1101/2020.08.21.262097

Figure Lengend Snippet: Generation of Cell-Specific Nrxn1 cKO Mice and Quantification of Neurexin Protein Levels (data complement those shown in figures 1-2). a-b, Breeding strategies to generate neuron- ( a ), and astrocyte-specific Nrxn1 cKOs ( b ). Note that these breeding strategies operate either with classical Nrxn1 cKO mice or with Nrxn1 conditional HA-knockin mice. All functional studies were performed with classical cKO mice, and conditional HA-knockin mice were only used for studies of the localization and modification of Nrxn1 ( Figs. 1 and 2 ; Extended Figs. 2 - 6 ). c-d, Effect of neuron- ( c ) and astrocyte-specific Nrxn1 cKOs ( d ) on mouse survival (left graphs) and mouse weight at weaning (right graphs). Numbers in bars represent number of mice examined. e-f, Representative immunoblots of neurexins ( e ) and quantifications of their levels ( f ) using a series of antibodies with different degrees of specificity. Total brain samples from neuron- and astrocyte-specific Nrxn1 cKO mice and littermate controls were examined. Three different antibodies raised against the conserved cytoplasmic domains of Nrxns were used to recognize pan-α-Nrxns (i.e. G394, ABN161, and AF870) and a single antibody was used to recognize pan-β-Nrxn (i.e. G393). Note that most antibodies recognize multiple neurexins, and not only Nrxn1. g, Schematic of Nrxn1 proteins produced in conditional Nrxn1 HA-knockin mice. Before Cre-recombination, Nrxn1 HA-knockin mice express HA-tagged Nrxn1; after Cre-recombination, HA-Nrxn1 is truncated and secreted, resulting in cKO-T mice. Because the HA epitope is knocked into the Nrxn1 coding sequence C-terminal to the LNS6 domain followed by loxP sites, Cre-mediated truncated causes production of secreted Nrxn1α, Nrxn1β, and Nrxn1γ fragments that include a C-terminal HA-epitope. These fragments manifest on immunoblots as tight bands that are slightly smaller than wild-type Nrxn1 species (LNS1–6, LNS1-6 domains; E, EGF-like domain). h-I , Quantitative immunoblot analysis of neurexins after neuron- ( h ) or astrocyte-specific Cre-recombination ( i ) in Nrxn1 HA-knockin mice creating truncated neurexin variants (left, representative immunoblots using HA-antibodies; right, quantifications of Nrxn1α, Nrxn1β, and Nrxn1γ). Note that owing to the truncated HA-tagged Nrxn1 fragment after Cre-recombination, the deletions do not abolish as much of the Nrxn1 signal as observed in standard Nrxn1 cKO mice analyzed by Nrxn1 antibodies (see Fig. 2 ). Numerical data are means ± SEM. n = indicated on all graphs except for body weight in c-d with 60 Nrxn1 FL/FL (cre-), 23 Nrxn1 wt/FL (cre+), and 28 Nrxn1 FL/FL (cre+) mice ( c , right); 36 Nrxn1 FL /FL (cre-) and 32 Nrxn1 FL /FL (cre+) mice ( d , right). Statistical significance was assessed with a chi²-test ( c and f , survival), one-way ANOVA with a Tukey’s post-hoc test ( c and f , body weight), and two-tailed unpaired t-test to controls (rest of f , h , i ), with * = p

Article Snippet: Primary AntibodiesThe following antibodies were used at the indicated concentrations (IHC-immunohistochemistry; ICC-immunocytochemistry; IB-immunoblot): purified anti-HA mouse (Biolegend Cat# 901501; 1:500 live surface ICC, 1:500 ICC, 1:500 IHC, 1:1000 IB), purified anti-HA mouse Alexa647-conjugated (Biolegend Cat# 682404; 1:500 IHC), anti-HA rabbit (Cell Signaling Cat#3724; 1:250 ICC), anti-Homer1 rabbit (Millipore Cat# ABN37; 1:1000 IHC) anti-Nrxn1 rabbit (Synaptic Systems Cat# 175103; 1:1000 IB), anti-pan-Nrxn rabbit (Frontier Institute Cat# AF870; 1:500 IB), anti-pan-Nrxn rabbit (homemade, G393; 1:500 IB), anti-pan-Nrxn rabbit (homemade, G394; 1:500 IB), anti-pan-Nrxn rabbit (Millipore Cat# ABN-161-l; 1:1000 IB), anti-laminin-alpha2 rat (Abcam Cat# ab11576; 1:5000 IHC), anti-GFAP mouse (Neuromab Cat# 75-240; 1:1000 IB, 1:1000 ICC, 1:500 IHC), anti-GFAP rabbit (Agilent Cat# 033401-2; 1:1000 ICC, 1:1000 IHC), anti-GFAP chicken (Encorbio Cat# CPCA-GFAP; 1:1000 ICC, 1:1000 IHC), anti-HS-stub 3G10 antibody mouse (Amsbio Cat# 370260-1; 1:1000 IB), anti-vGluT1 (Homemade YZ6089; 1:1000 IHC, 1:1000 IB), anti-MAP2 mouse (Sigma Cat# M1406; 1:1000 IHC), anti-NeuN mouse (Millipore Cat# MAB377; 1:1000 IHC), anti-NeuN rabbit (1:1000 IHC), anti-ß-actin mouse (Sigma Cat#A1978; 1:3000 IB), anti-Synapsins rabbit (Homemade YZ6078; 1:500 ICC, 1 :1000 IB), anti-Flag rat (Sigma Cat# SAB4200071; 1:500 surface ICC), anti-Myc rat (Abcam Cat# ab206486; 1:500 surface ICC), anti-VGAT guinea pig (Synaptic Systems Cat# 131005; 1:500 IHC), anti-GluN1 mouse (Synaptic Systems Cat# 114011; 1:1000 IB), anti-GluN2B mouse (Neuromab Cat# 75-101; 1:1000 IB), anti-GluR1 rabbit (Millipore Cat# Ab1504; 1:1000 IB), anti-GluR2 mouse (Neuromab Cat# 75-002; 1:1000 IB), anti-GluR4 (Millipore Cat# Ab1508; 1:1000 IB), anti-PSD-95 mouse (Neuromab Cat# 75-028; 1:1000 IB), anti-GRIP mouse (1:1000 IB), anti-Gad67 mouse (Millipore Cat# mab5406B; 1:1000 IB), anti-SNAP25 rabbit (Homemade P913; 1:500 IB), anti-Nlgn1 mouse (Synaptic Systems Cat# 129111; 1:1000 IB), anti-Nlgn2 rabbit (Synaptic Systems Cat# 129203; 1:1000 IB), anti-Nlgn3 mouse (Synaptic Systems Cat# 129311; 1:2000 IB), anti-GluD1 rabbit (Frontier Institute Cat# GluD1C-Rb-Af1390; 1:2000 IB), anti-CASK mouse (NeuroMab Cat# 75-000; 1:1000 IB). anti-Kir4.1 rabbit (Alomone Cat# APC-035; 1:2000 IB), anti-Glt1 guinea pig (Millipore Cat# AB1783; 1:1000 IB).

Techniques: Mouse Assay, Knock-In, Functional Assay, Modification, Western Blot, Produced, Sequencing, Two Tailed Test