rabbit anti nt3  (Alomone Labs)

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Mutation of phosphorylated serines does not affect subcellular localization of Kv7.1. Alanine ( A ) and aspartate ( B ) mutants of the identified serine phosphorylation sites were coexpressed with the ER marker pDsRed2-ER in MDCK cells. The cells were stained

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Mutagenesis, Marker, Staining

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Summary of proteomics results for Kv7.1. A : topological representation of the Kv7.1 channel, including the amino acid sequence. The amino acid sequences highlighted in blue were covered in the mass spectrometry experiments, and the identified phosphorylation

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Sequencing, Mass Spectrometry

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Inhibition of PKA leads to internalization and possibly lysosomal degradation of Kv7.1.

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Inhibition

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Kv7.1 accumulates intracellularly upon exposure to PKA inhibitors KT-5720 and myristoylated protein kinase A inhibitor 14–22 amide. A : MDCK cells stably expressing Kv7.1 were subjected to a calcium switch for 24 h (control) followed by treatment

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Stable Transfection, Expressing

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Kv7.1 codistributes with late endosomal/lysosomal markers in response to H-89 treatment. A : confocal scans of MDCK-Kv7.1 cells treated with 20 μM H-89 for 3 h and colabeled with the late endosomal/lysosomal marker LAMP-2. Scale bar = 10 ( top )

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Marker

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Small-interfering RNA (siRNA) knockdown of Nedd4-2 protects Kv7.1 from internalization upon PKA inhibition. A : MDCK cells stably expressing Kv7.1 were transiently transfected with siRNA targeting Nedd4-2 or with noncoding control siRNA. Enhanced GFP (eGFP)

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Small Interfering RNA, Inhibition, Stable Transfection, Expressing, Transfection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Kv7.1-YA localization is unaffected by PKA inhibition. MDCK cells stably expressing the Kv7.1-YA mutant were treated with 20 μM H-89 for 3–5 h. A : confocal scans of MDCK-Kv7.1-YA cells before (0 h) and 3 (3 h H-89) and 5 (5 h H-89) h after

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Inhibition, Stable Transfection, Expressing, Mutagenesis

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Kv7.1 redistribution is not caused by increased Nedd4-2 binding or ubiquitinylation. A : representative Western blot showing the effects of 20 μM H-89 (3 h exposure) and 50 μM forskolin (1 h exposure) on the abundance of S 468 -phosphorylated

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Binding Assay, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Kv7.1 accumulates intracellularly upon inhibition of protein kinase A (PKA). Madin-Darby canine kidney (MDCK) cells stably expressing Kv7.1 were subjected to a calcium switch for 24 h followed by treatment with 20 μM H-89 for 3–5 h. A

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Inhibition, Stable Transfection, Expressing

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Intracellular accumulation in response to PKA inhibition is specific for Kv7.1. MDCK cells were transfected with Kv7.4 and allowed to polarize before being subjected to either 20 μM H-89 or 10 μM KT-5720 treatments for 3 h. As demonstrated,

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Inhibition, Transfection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: PKA activation promotes Kv7.1 surface expression in polarized MDCK cells. MDCK cells stably expressing Kv7.1 were grown to confluence and allowed to polarize. The cells were then treated with 50 μM forskolin for 1 h. Cells were fixed before and

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Activation Assay, Expressing, Stable Transfection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

doi: 10.1152/ajpcell.00383.2014

Figure Lengend Snippet: Mimicked phosphorylation of T470 results in reduced membrane expression of Kv7.1, but Kv7.1-T470A is still endocytosed in response to PKA inhibition. Kv7.1-T470A and Kv7.1-T470D mutants were coexpressed with DsRed2-ER in MDCK cells, which were allowed

Article Snippet: The precleared lysate was subsequently incubated with 3 μg of rabbit anti-Kv7.1 antibody (Alomone Labs), or, as a control, 3 μg normal rabbit IgG (Santa Cruz Biotechnology) for 1 h at 4°C.

Techniques: Expressing, Inhibition

Journal: Pain

Article Title: Epac–protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation

doi: 10.1097/j.pain.0000000000000547

Figure Lengend Snippet: Go6976 reduces α,β-meATP–elicited flinch nocifensive responses in complete Freund adjuvant–treated rats, and PKCβ1 is not expressed in P2X3R-containing dorsal root ganglia (DRG) neurons. (A) In saline control rats,

Article Snippet: Antibodies used were mouse anti-Epac1, anti-Epac2 (1:1000; Cell Signaling, Danvers, MA), mouse anti-PKCε (1:1000; Santa Cruz), rabbit anti-pPKCε (1:1000; Santa Cruz), and rabbit anti-P2X3R (1:2000; Alomone Labs). β-Actin, probed with anti-β-actin (1:10,000; Chemicon, Temeculla, CA), or the neuronal marker, β-tubulin, probed with anti β-tubulin antibody (1:2000; Santa Cruz), was used as loading control.

Techniques:

Journal: Pain

Article Title: Epac–protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation

doi: 10.1097/j.pain.0000000000000547

Figure Lengend Snippet: Phosphorylated PKC (pPKC) isoform and P2X3R expression in dorsal root ganglia slices prepared from complete Freund adjuvant–treated rats. Upper: pPKCα and P2X3Rs were expressed only in small or medium cells. pPKCα labels were found

Article Snippet: Antibodies used were mouse anti-Epac1, anti-Epac2 (1:1000; Cell Signaling, Danvers, MA), mouse anti-PKCε (1:1000; Santa Cruz), rabbit anti-pPKCε (1:1000; Santa Cruz), and rabbit anti-P2X3R (1:2000; Alomone Labs). β-Actin, probed with anti-β-actin (1:10,000; Chemicon, Temeculla, CA), or the neuronal marker, β-tubulin, probed with anti β-tubulin antibody (1:2000; Santa Cruz), was used as loading control.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Induction of detrusor underactivity by extensive vascular endothelial damages of iliac arteries in a rat model and its pathophysiology in the genetic levels

doi: 10.1038/s41598-019-52811-4

Figure Lengend Snippet: Repression of P2rx1 by progressive vascular endothelial damage. ( a – c ) RQ-PCR ( a ) and western blot ( b , c ) analysis of the expressions of the cholinergic receptor muscarinic 2 ( Chrm2 ), cholinergic receptor muscarinic 3 ( Chrm3 ), and purinergic receptor P2X 1 ( P2rx1 ) genes in the bladders of rats in the indicated groups. Expression levels of the indicated transcripts are presented as % Gapdh . For western blot analysis, β-actin was used as a loading control, and the expression levels of the indicated proteins were normalized to the β-actin value. ( d , e ) Representative images ( d ) and quantification analysis ( e ) of the immunohistochemical staining for the P2rx1 protein (original magnification ×200, scale bar = 200 μm). All quantification results are presented as mean ± SEM. *p

Article Snippet: Protein levels were assessed from 50 μg of extracts separated using 10% SDS-PAGE gels by probing with antibodies specific to P2rx1 (APR-001, Alomone Labs), Chrm2 (AMR-002, Alomone Labs,), Chrm3 (AMR-006, Alomone Labs, Israel), α-SMA (ab5694, Rabbit, Abcam), vimentin (sc-6260, Santa Cruz Biotechnology Inc., CA), and β-actin (Sigma-Aldrich).

Techniques: Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemistry, Staining

Journal: The Journal of Physiology

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: Anti-ClC-3 Ab dialysis abolishes expressed I gpClC-3 in NIH/3T3 cells A , phase contrast (left panel) and fluorescence micrographs (right panel) of NIH/3T3 cell transfected with gpClC-3-GFP. B , representative I gpClC-3 recorded from GFP-positive NIH/3T3 cells (as shown in A ) under isotonic, hypotonic and hypertonic conditions over the range -100 to +120 mV. C , effects of anti-ClC-3 Ab on I gpClC-3 at ±80 mV when pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). D , mean current densities recorded from untransfected NIH/3T3 cells (blue, n = 38), gpClC-3-GFP-transfected cells dialysed with standard intracellular pipette solutions (green, n = 9), preabsorbed anti-ClC-3 Ab (black, n = 4), and anti-ClC-3 Ab alone (red, n = 9).

Article Snippet: We first demonstrate that intracellular dialysis with a commercially available antibody which targets a specific 70 amino acid epitope on the carboxyl terminus of rat ClC-3 (anti-ClC-3 Ab) abolishes expressed I gpClC-3 in transfected NIH/3T3 cells, and then demonstrate that intracellular dialysis or injection of anti-ClC-3 Ab into native cardiac and vascular smooth muscle myocytes and Xenopus oocytes abolishes or significantly attenuates native VSOAC function.

Techniques: Fluorescence, Transfection, Transferring

Journal: The Journal of Physiology

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: Anti-ClC-3 Ab abolishes native VSOAC currents in canine PASMCs A , time course of VSOAC currents from two PASMCs dialysed with either anti-ClC-3 Ab (5 μg ml −1 ) preabsorbed with antigen (50 μg ml −1 ; ○), or anti-ClC-3 Ab alone (5 μg ml −1 ; •). Inset: Western blot analysis of native ClC-3 expression in isolated canine PASMCs. B , mean current densities in cells dialysed with either standard intracellular solutions ( n = 11), preabsorbed anti-ClC-3 Ab ( n = 4) or anti-ClC-3 Ab alone ( n = 5).

Article Snippet: We first demonstrate that intracellular dialysis with a commercially available antibody which targets a specific 70 amino acid epitope on the carboxyl terminus of rat ClC-3 (anti-ClC-3 Ab) abolishes expressed I gpClC-3 in transfected NIH/3T3 cells, and then demonstrate that intracellular dialysis or injection of anti-ClC-3 Ab into native cardiac and vascular smooth muscle myocytes and Xenopus oocytes abolishes or significantly attenuates native VSOAC function.

Techniques: Western Blot, Expressing, Isolation

Journal: The Journal of Physiology

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: Anti-ClC-3 Ab dialysis abolishes native VSOAC currents in guinea-pig cardiac myocytes A , native VSOAC currents at ±80 mV in myocytes exposed to isotonic and hypotonic solutions. Aa , pipette solutions contained preabsorbed anti-ClC-3 Ab (control, ○) or anti-ClC-3 Ab alone (▪). Ab , mean current densities for preabsorbed anti-ClC-3 Ab (□, n = 3) or anti-ClC-3 Ab alone (▪, n = 5) at times indicated. B, same as A , except cells were preswelled by pre-exposure to hypotonic bath solutions prior to membrane rupture and dialysis; preabsorbed anti-ClC-3 Ab (□, n = 4), and anti-ClC-3 Ab alone (▪, n = 5).

Article Snippet: We first demonstrate that intracellular dialysis with a commercially available antibody which targets a specific 70 amino acid epitope on the carboxyl terminus of rat ClC-3 (anti-ClC-3 Ab) abolishes expressed I gpClC-3 in transfected NIH/3T3 cells, and then demonstrate that intracellular dialysis or injection of anti-ClC-3 Ab into native cardiac and vascular smooth muscle myocytes and Xenopus oocytes abolishes or significantly attenuates native VSOAC function.

Techniques: Transferring

Journal: The Journal of Physiology

Article Title: Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

doi: 10.1111/j.1469-7793.2001.0437i.x

Figure Lengend Snippet: Anti-ClC-3 Ab injection inhibits native VSOAC currents in Xenopus laevis oocytes A , Western blot analysis of native ClC-3 expression in oocytes. B , 4-phorbol 12,13-dibutyrate (PDBu) inhibition of native VSOAC currents in oocytes (-100 to +120 mV). C, time course of VSOAC activation in response to hypotonic bath solutions from a non-injected control oocyte (○) and an oocyte injected with anti-ClC-3 Ab (15 μg ml -1 ; •) at time shown. Cells were held at -30 mV for 30 ms, hyperpolarized to -100 mV for 210 ms and then depolarized to +100 mV for 210 ms, repetitively at 2 Hz. D, mean current densities from control oocytes ( n = 10), oocytes injected with anti-ClC-3 Ab alone (15 μg ml -1 ) ( n = 4) or injected with preabsorbed anti-ClC-3 Ab (150 μg ml -1 ) ( n = 4). Peak current densities were measured after 5 min exposure to isotonic solutions, and after 85 min exposure to hypotonic solutions.

Article Snippet: We first demonstrate that intracellular dialysis with a commercially available antibody which targets a specific 70 amino acid epitope on the carboxyl terminus of rat ClC-3 (anti-ClC-3 Ab) abolishes expressed I gpClC-3 in transfected NIH/3T3 cells, and then demonstrate that intracellular dialysis or injection of anti-ClC-3 Ab into native cardiac and vascular smooth muscle myocytes and Xenopus oocytes abolishes or significantly attenuates native VSOAC function.

Techniques: Injection, Western Blot, Expressing, Inhibition, Activation Assay, Mass Spectrometry