anti atpase na k β2  (Bioss)

Journal: International Journal of Molecular Sciences

Article Title: Deficiency of Thyroid Hormone Reduces Voltage-Gated Na + Currents as Well as Expression of Na + /K + -ATPase in the Mouse Hippocampus

doi: 10.3390/ijms23084133

Figure Lengend Snippet: Effects of congenital hypothyroidism on sodium potassium ATPase α and β subunit expression. Protein levels of Na + /K + -ATPases ( a ) α1, α2, and α3 isoforms, and ( b ) β1 and β2 isoforms from P19–P21 old Pax8 −/− (light gray), Pax8 +/− (dark gray), and Pax8 +/+ (white) mice. Exemplary immunoblotting bands are shown below bar charts. β tubulin was used as internal control. Bars represent means ± SEM. Data from individual animals depicted as diamonds. Statistically significant differences marked with * ( p < 0.05) or ** ( p < 0.01), n = 3–4 for each condition.

Article Snippet: Membranes were blocked in 5% dry milk powder in Tris-buffered saline (TBS) and incubated overnight at 4 °C with primary antibodies against Na + /K + -ATPase isoforms [anti-Na + /K + -ATPase α1, Millipore (05–369) dilution 1:2500; anti-Na + /K + -ATPases α2, Millipore (07–674) dilution 1:2500; anti-Na + /K + -ATPases α3, Millipore (06–172) dilution 1:2500; anti-Na + /K + -ATPases β1, Millipore (06–170) dilution1:2500; anti-Na + /K + -ATPases β2 Millipore (06–171) dilution 1:2500], and anti β-tubulin (Sigma/T4026, dilution 1:5000).

Techniques: Expressing, Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: Glutamate Transporter Coupling to Na,K-ATPase

doi: 10.1523/JNEUROSCI.1081-09.2009

Figure Lengend Snippet: Colocalization of GLAST and GFAP with α2 Na,K-ATPase in the rat cerebellar cortex. A, Immunocytochemical distribution of α2 Na,K-ATPase in Bergmann glia processes (arrowhead) and Purkinje cell bodies (arrow) in the cerebellar cortex. B, C, GFAP expression (B) and merged image of A and B (C) showing colocalization in the processes of the Bergmann glia (arrowhead) but not in Purkinje cells (arrow). D–F, The colocalization of α2 Na,K-ATPase (D) and GLAST (using anti-GLAST mouse monoclonal antibody; E) in the Bergmann glia processes (arrowheads) is depicted in the merged image shown in F. G, Negative control with no primary antibody. H, I, In situ hybridization analysis of GLAST (H) and α2 Na,K-ATPase mRNA (I) in the mouse cerebellum (from the Allen Brain Atlas). GL, Granule cell layer; ML, molecular layer; PL, Purkinje cell layer of the cerebellar cortex. Scale bars, 50 μm.

Article Snippet: The blots were then washed three times with wash buffer (0.01 m Tris-HCl, 0.15 m NaCl, and 0.2% Tween 20, pH 8.0) at room temperature, followed by incubation for 2 h with the primary antibodies as follows: anti-GLAST [1:1000, rabbit polyclonal (Santa Cruz Biotechnology) or 1:500, mouse monoclonal antibody (Novocastra Laboratories)], anti-GLT-1 [1:2000, rabbit polyclonal raised against the sequence N(556)GKSADCSVEEEPWKREK(573) of rat GLT-1 (Affinity BioReagents)], nonselective anti-α Na,K-ATPase (1:2000, mouse monoclonal; Affinity BioReagents), anti-α1 Na,K-ATPase (6F, 1:500, mouse monoclonal raised against N-terminal amino acids 27–55; University of Iowa, Iowa City, IA), anti-α2 Na,K-ATPase (1:2000, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-α3 Na,K-ATPase (1:2000, mouse monoclonal clone XVIF9-G10; Affinity BioReagents), anti-β2 Na,K-ATPase (1:1000, mouse monoclonal clone 35; BD Biosciences), and anti-c- myc (1:1000, mouse monoclonal clone 9E10 raised against amino acids 408–437 of the leucine zipper region of human Myc; Millipore).

Techniques: Expressing, Negative Control, In Situ Hybridization

Journal: The Journal of Neuroscience

Article Title: Glutamate Transporter Coupling to Na,K-ATPase

doi: 10.1523/JNEUROSCI.1081-09.2009

Figure Lengend Snippet: Colocalization of α2 and α3 Na,K-ATPase with GLT-1 in the CA1 region of the rat hippocampus. A, Immunocytochemical distribution of α2 Na,K-ATPase visualized with a tetramethylrhodamine isothiocyanate-conjugated anti-rabbit secondary. B, C, GLT-1b expression visualized with FITC-conjugated anti-mouse secondary antibody (B) and merged portion of images of A and B within boxed region indicated in B (C) showing colocalization in astrocyte processes (arrows). D–F, The colocalization of GLT-1a (rabbit polyclonal; D) with α3 Na,K-ATPase (mouse monoclonal; E) primarily in the pyramidal cell layer is depicted in the merged image shown in F. G–I, The colocalization of GLT-1a (G) and the astrocyte marker GFAP (mouse monoclonal; H) is shown in the merged image in I (example indicated by arrow). Asterisks indicate pyramidal cell layer. Scale bars, 50 μm.

Article Snippet: The blots were then washed three times with wash buffer (0.01 m Tris-HCl, 0.15 m NaCl, and 0.2% Tween 20, pH 8.0) at room temperature, followed by incubation for 2 h with the primary antibodies as follows: anti-GLAST [1:1000, rabbit polyclonal (Santa Cruz Biotechnology) or 1:500, mouse monoclonal antibody (Novocastra Laboratories)], anti-GLT-1 [1:2000, rabbit polyclonal raised against the sequence N(556)GKSADCSVEEEPWKREK(573) of rat GLT-1 (Affinity BioReagents)], nonselective anti-α Na,K-ATPase (1:2000, mouse monoclonal; Affinity BioReagents), anti-α1 Na,K-ATPase (6F, 1:500, mouse monoclonal raised against N-terminal amino acids 27–55; University of Iowa, Iowa City, IA), anti-α2 Na,K-ATPase (1:2000, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-α3 Na,K-ATPase (1:2000, mouse monoclonal clone XVIF9-G10; Affinity BioReagents), anti-β2 Na,K-ATPase (1:1000, mouse monoclonal clone 35; BD Biosciences), and anti-c- myc (1:1000, mouse monoclonal clone 9E10 raised against amino acids 408–437 of the leucine zipper region of human Myc; Millipore).

Techniques: Expressing, Marker

Journal: The Journal of Neuroscience

Article Title: Glutamate Transporter Coupling to Na,K-ATPase

doi: 10.1523/JNEUROSCI.1081-09.2009

Figure Lengend Snippet: Copurification of GLAST and α Na,K-ATPase from adult rat cerebellum by tandem DEAE anion exchange and ouabain affinity chromatography. Chromatography fractions were analyzed on Western blots (WB) probed with a nonselective α Na,K-ATPase (NKA) antibody (left), an α2 Na,K-ATPase-specific antibody (middle), and a GLAST antibody (right). A–C, The α subunits of Na,K-ATPase (seen at 100 kDa in A, B) and the monomeric and dimeric forms of GLAST seen at 65 and 130 kDa (C) were detected in the final eluate from the ouabain affinity column (OUA eluate). Sol CB, Crude solubilized cerebellar membranes; OUA UR, unretained fraction from ouabain affinity column.

Article Snippet: The blots were then washed three times with wash buffer (0.01 m Tris-HCl, 0.15 m NaCl, and 0.2% Tween 20, pH 8.0) at room temperature, followed by incubation for 2 h with the primary antibodies as follows: anti-GLAST [1:1000, rabbit polyclonal (Santa Cruz Biotechnology) or 1:500, mouse monoclonal antibody (Novocastra Laboratories)], anti-GLT-1 [1:2000, rabbit polyclonal raised against the sequence N(556)GKSADCSVEEEPWKREK(573) of rat GLT-1 (Affinity BioReagents)], nonselective anti-α Na,K-ATPase (1:2000, mouse monoclonal; Affinity BioReagents), anti-α1 Na,K-ATPase (6F, 1:500, mouse monoclonal raised against N-terminal amino acids 27–55; University of Iowa, Iowa City, IA), anti-α2 Na,K-ATPase (1:2000, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-α3 Na,K-ATPase (1:2000, mouse monoclonal clone XVIF9-G10; Affinity BioReagents), anti-β2 Na,K-ATPase (1:1000, mouse monoclonal clone 35; BD Biosciences), and anti-c- myc (1:1000, mouse monoclonal clone 9E10 raised against amino acids 408–437 of the leucine zipper region of human Myc; Millipore).

Techniques: Copurification, Affinity Chromatography, Chromatography, Western Blot, Affinity Column

Journal: The Journal of Neuroscience

Article Title: Glutamate Transporter Coupling to Na,K-ATPase

doi: 10.1523/JNEUROSCI.1081-09.2009

Figure Lengend Snippet: Coimmunoprecipitation of GLAST and GLT-1 with α2 and α3 Na,K-ATPase in rat brain. A, B, Western blots (WB) of α2 (A) and α3 Na,K-ATPase (B) showing immunoprecipitation (IP) from rat forebrain (FB) and/or cerebellum (CB) using anti-α2 and α3 Na,K-ATPase antibodies. C, Western blot showing coimmunoprecipitation of GLT-1 with α2 and α3 Na,K-ATPase from rat forebrain. D, Coimmunoprecipitation of GLAST with α2 Na,K-ATPase in rat cerebellum. SOL., Crude solubilized membranes from forebrain or cerebellum used as a positive control for the antibody on the Western blot; −AB, negative control excluding antibody from the immunoprecipitation.

Article Snippet: The blots were then washed three times with wash buffer (0.01 m Tris-HCl, 0.15 m NaCl, and 0.2% Tween 20, pH 8.0) at room temperature, followed by incubation for 2 h with the primary antibodies as follows: anti-GLAST [1:1000, rabbit polyclonal (Santa Cruz Biotechnology) or 1:500, mouse monoclonal antibody (Novocastra Laboratories)], anti-GLT-1 [1:2000, rabbit polyclonal raised against the sequence N(556)GKSADCSVEEEPWKREK(573) of rat GLT-1 (Affinity BioReagents)], nonselective anti-α Na,K-ATPase (1:2000, mouse monoclonal; Affinity BioReagents), anti-α1 Na,K-ATPase (6F, 1:500, mouse monoclonal raised against N-terminal amino acids 27–55; University of Iowa, Iowa City, IA), anti-α2 Na,K-ATPase (1:2000, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-α3 Na,K-ATPase (1:2000, mouse monoclonal clone XVIF9-G10; Affinity BioReagents), anti-β2 Na,K-ATPase (1:1000, mouse monoclonal clone 35; BD Biosciences), and anti-c- myc (1:1000, mouse monoclonal clone 9E10 raised against amino acids 408–437 of the leucine zipper region of human Myc; Millipore).

Techniques: Western Blot, Immunoprecipitation, Positive Control, Negative Control

Journal: The Journal of Neuroscience

Article Title: Glutamate Transporter Coupling to Na,K-ATPase

doi: 10.1523/JNEUROSCI.1081-09.2009

Figure Lengend Snippet: Inhibition of [3H]d-aspartate, [3H]l-glutamate, and rubidium-86 uptake by ouabain in synaptosomes from rat forebrain and cerebellum. A, Western blot analysis of forebrain (FB) and cerebellar (CB) synaptosomes demonstrating expression of GLAST and GLT-1. B, Western blot analysis of forebrain (FB) and cerebellar (CB) synaptosomes demonstrating expression of α1–α3 isoforms of Na,K-ATPase. C, D, Results of d-aspartate and l-glutamate uptake assays in forebrain (C) and cerebellar (D) synaptosomes expressed as percentage of control [sodium-containing buffer, (+) Na]. Inhibition of both d-aspartate and l-glutamate uptake was observed with the nonselective EAAT inhibitor TBOA (200 μm), the GLT-1-selective inhibitors DHK (200 μm) and WAY213613 (WAY; 1 μm), and ouabain (OUA; 1 mm). Each column represents the mean and SEM of three to seven experiments. (−) Na, Omission of sodium from the assay buffer. E, Left, Concentration-dependent inhibition of rubidium-86 uptake by ouabain in rat forebrain (▴; IC50 of 1.36 × 10−4 m) synaptosomes. The results are expressed as percentage of control [sodium-containing buffer, (+) Na]. Each point represents the mean ± SEM of six experiments done in triplicate. E, Right, Concentration-dependent inhibition of [3H]d-aspartate uptake by ouabain in rat forebrain (▴; IC50 of 1.62 × 10−5 m) and cerebellar (■; IC50 of 2.61 × 10−5 m) synaptosomes. Results are expressed as percentage of control [sodium-containing buffer, (+) Na]. Each point represents the average of two experiments done in triplicate. p values compare test condition with controls without drug.

Article Snippet: The blots were then washed three times with wash buffer (0.01 m Tris-HCl, 0.15 m NaCl, and 0.2% Tween 20, pH 8.0) at room temperature, followed by incubation for 2 h with the primary antibodies as follows: anti-GLAST [1:1000, rabbit polyclonal (Santa Cruz Biotechnology) or 1:500, mouse monoclonal antibody (Novocastra Laboratories)], anti-GLT-1 [1:2000, rabbit polyclonal raised against the sequence N(556)GKSADCSVEEEPWKREK(573) of rat GLT-1 (Affinity BioReagents)], nonselective anti-α Na,K-ATPase (1:2000, mouse monoclonal; Affinity BioReagents), anti-α1 Na,K-ATPase (6F, 1:500, mouse monoclonal raised against N-terminal amino acids 27–55; University of Iowa, Iowa City, IA), anti-α2 Na,K-ATPase (1:2000, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-α3 Na,K-ATPase (1:2000, mouse monoclonal clone XVIF9-G10; Affinity BioReagents), anti-β2 Na,K-ATPase (1:1000, mouse monoclonal clone 35; BD Biosciences), and anti-c- myc (1:1000, mouse monoclonal clone 9E10 raised against amino acids 408–437 of the leucine zipper region of human Myc; Millipore).

Techniques: Inhibition, Western Blot, Expressing, Concentration Assay

Journal: The Journal of Neuroscience

Article Title: Glutamate Transporter Coupling to Na,K-ATPase

doi: 10.1523/JNEUROSCI.1081-09.2009

Figure Lengend Snippet: Expression of glutamate transporters and Na,K-ATPase subtypes and drug effects on d-aspartate uptake in rat astrocytes. A, Western blot analysis of cultured astrocytes (Ast; 11–14 d in vitro) showed expression of GLAST (65 kDa) but not GLT-1. B, Western blots of astrocytes demonstrating expression of α1, α2, and β2 subunits of Na,K-ATPase (NKA; 100, 100, and 45 kDa, respectively) but not the α3 subunit. Forebrain (FB) and cerebellar (CB) membranes were used as positive controls. C, Summary of drug effects on [3H]d-aspartate uptake in cultured astrocytes expressed as percentage of control [sodium-containing buffer only, (+) Na]. Each bar represents the mean ± SEM of three to five experiments. DHK (200 μm) and WAY213613 (1 μm) did not significantly inhibit uptake, whereas TBOA (200 μm) and ouabain (OUA) at >50 μm significantly inhibited uptake. Ouabain at 1 μm significantly stimulated d-aspartate uptake. *p < 0.05, **p < 0.001 compared with control (+Na).

Article Snippet: The blots were then washed three times with wash buffer (0.01 m Tris-HCl, 0.15 m NaCl, and 0.2% Tween 20, pH 8.0) at room temperature, followed by incubation for 2 h with the primary antibodies as follows: anti-GLAST [1:1000, rabbit polyclonal (Santa Cruz Biotechnology) or 1:500, mouse monoclonal antibody (Novocastra Laboratories)], anti-GLT-1 [1:2000, rabbit polyclonal raised against the sequence N(556)GKSADCSVEEEPWKREK(573) of rat GLT-1 (Affinity BioReagents)], nonselective anti-α Na,K-ATPase (1:2000, mouse monoclonal; Affinity BioReagents), anti-α1 Na,K-ATPase (6F, 1:500, mouse monoclonal raised against N-terminal amino acids 27–55; University of Iowa, Iowa City, IA), anti-α2 Na,K-ATPase (1:2000, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-α3 Na,K-ATPase (1:2000, mouse monoclonal clone XVIF9-G10; Affinity BioReagents), anti-β2 Na,K-ATPase (1:1000, mouse monoclonal clone 35; BD Biosciences), and anti-c- myc (1:1000, mouse monoclonal clone 9E10 raised against amino acids 408–437 of the leucine zipper region of human Myc; Millipore).

Techniques: Expressing, Western Blot, Cell Culture, In Vitro

Journal: The Journal of Neuroscience

Article Title: Glutamate Transporter Coupling to Na,K-ATPase

doi: 10.1523/JNEUROSCI.1081-09.2009

Figure Lengend Snippet: Expression of glutamate transporters and Na,K-ATPase and coimmunoprecipitation of GLAST and α2 Na,K-ATPase from α2β2GLAST-transfected HEK-293T cells. A, Western blot (WB) analysis of mock-transfected HEK-293T cells (Mock) showing expression of α1 andα3 Na,K-ATPase (100 kDa) and GLAST (monomer at 65 kDa) and the absence of α2 Na,K-ATPase and little or no GLT-1. Rat forebrain (FB) membranes were used as positive controls. B, Antibodies selective for α2 Na,K-ATPase and GLAST were used to immunoprecipitate the respective proteins from HEK-293T cells cotransfected with GLAST, α2, and β2 Na,K-ATPase cDNAs. The samples were analyzed by Western blots probed with GLAST (left) and α2 Na,K-ATPase-specific (right) antibodies. The α2 subunit of Na,K-ATPase (seen at 100 kDa) coimmunoprecipitated using the GLAST antibody, and, conversely, GLAST (dimer seen at 130 kDa) coimmunoprecipitated using the α2 Na,K-ATPase-specific antibody. SOL., Crude solubilized HEK-293T cells cotransfected with GLAST, α2, and β2 Na,K-ATPase; −AB, negative control excluding antibody in the immunoprecipitation step.

Article Snippet: The blots were then washed three times with wash buffer (0.01 m Tris-HCl, 0.15 m NaCl, and 0.2% Tween 20, pH 8.0) at room temperature, followed by incubation for 2 h with the primary antibodies as follows: anti-GLAST [1:1000, rabbit polyclonal (Santa Cruz Biotechnology) or 1:500, mouse monoclonal antibody (Novocastra Laboratories)], anti-GLT-1 [1:2000, rabbit polyclonal raised against the sequence N(556)GKSADCSVEEEPWKREK(573) of rat GLT-1 (Affinity BioReagents)], nonselective anti-α Na,K-ATPase (1:2000, mouse monoclonal; Affinity BioReagents), anti-α1 Na,K-ATPase (6F, 1:500, mouse monoclonal raised against N-terminal amino acids 27–55; University of Iowa, Iowa City, IA), anti-α2 Na,K-ATPase (1:2000, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-α3 Na,K-ATPase (1:2000, mouse monoclonal clone XVIF9-G10; Affinity BioReagents), anti-β2 Na,K-ATPase (1:1000, mouse monoclonal clone 35; BD Biosciences), and anti-c- myc (1:1000, mouse monoclonal clone 9E10 raised against amino acids 408–437 of the leucine zipper region of human Myc; Millipore).

Techniques: Expressing, Transfection, Western Blot, Negative Control, Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: Glutamate Transporter Coupling to Na,K-ATPase

doi: 10.1523/JNEUROSCI.1081-09.2009

Figure Lengend Snippet: Structural and functional aspects of glutamate transporter and Na,K-ATPase interactions. A, Depictions of the crystal structures of the pig Na,K-ATPase and a glutamate transporter from Pyrococcus horikoshii (graphics taken from Morth et al., 2007 and Yernool et al., 2004, respectively). B, Hypothetical model of the juxtaposition of glutamate (Glu) transporters with Na,K-ATPase in which only one of the subunits within the trimeric structure of the transporter is associated with Na,K-ATPase.

Article Snippet: The blots were then washed three times with wash buffer (0.01 m Tris-HCl, 0.15 m NaCl, and 0.2% Tween 20, pH 8.0) at room temperature, followed by incubation for 2 h with the primary antibodies as follows: anti-GLAST [1:1000, rabbit polyclonal (Santa Cruz Biotechnology) or 1:500, mouse monoclonal antibody (Novocastra Laboratories)], anti-GLT-1 [1:2000, rabbit polyclonal raised against the sequence N(556)GKSADCSVEEEPWKREK(573) of rat GLT-1 (Affinity BioReagents)], nonselective anti-α Na,K-ATPase (1:2000, mouse monoclonal; Affinity BioReagents), anti-α1 Na,K-ATPase (6F, 1:500, mouse monoclonal raised against N-terminal amino acids 27–55; University of Iowa, Iowa City, IA), anti-α2 Na,K-ATPase (1:2000, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-α3 Na,K-ATPase (1:2000, mouse monoclonal clone XVIF9-G10; Affinity BioReagents), anti-β2 Na,K-ATPase (1:1000, mouse monoclonal clone 35; BD Biosciences), and anti-c- myc (1:1000, mouse monoclonal clone 9E10 raised against amino acids 408–437 of the leucine zipper region of human Myc; Millipore).

Techniques: Functional Assay