Journal: Science advances

Article Title: ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids.

doi: 10.1126/sciadv.adi3647

Figure Lengend Snippet: Fig. 1. ATP1A3 is a neuron-specific EV marker candidate besides NCAM1 and L1CAM and associated with EVs in human brain tissues and biofluids at a single- particle level. (A) Experimental design for evaluating whether NDEV marker candidates (ATP1A3, NCAM1, and L1CAM) is associated with EVs. UC, ultracentrifugation; SU- GC, sucrose gradient centrifugation; SEC, size exclusion chromatography. (B) Transmission electron micrograph showing immunogold labeling of EVs isolated from iNeuron, brain, CSF, and plasma stained with anti-CD9 antibody (10 nm) and anti-ATP1A3, NCAM1, and L1CAM antibody (5 nm). Scale bars, 100 nm. (C) Representative fluorescent images of iNeuron-EV detected by fluorescent-conjugated antibodies (red: ATP1A3-AF647, NCAM1-AF647 or L1CAM-AF647; blue: CD9/81C/63-AF488) on Exoview chip CD63 capture spots. The round circle in magnified images masks costained particles with tetraspanins. Scale bars, 25 and 5 μm. (D) Percentages of ATP1A3, NCAM1, and L1CAM fluorescent particle counts relative to total particles in iNeuron derived EVs (n = 2 biological replicates) on Exoview chip tetraspanins capture spots. (E) Representative fluorescent images of brain- and CSF-EV detected by fluorescent-conjugated antibodies (green: ATP1A3-AF555; red: NCAM1-AF647 or L1CAM-AF647; blue: CD9/81/63-AF488) on Exoview chip CD9 capture spots. The round circle in magnified images masks co-stained particles with tetraspanins. Scale bars, 25 and 5 μm. (F) Percentages of ATP1A3, NCAM1, and L1CAM fluorescent particle counts relative to total particles in brain (n = 2) and CSF (n = 2) derived EVs on Exoview chip tetraspanins capture spots.

Article Snippet: In our experiment, EV antibodies cocktail (EV-TC-AB-01, NanoView Biosciences) containing AF488-conjugated anti-CD63, anti-CD81, and anti-CD9 antibodies, AF647-conjugated anti-ATP1A3 antibody (NBP299238AF647, Novus Biologicals), AF647-conjugated anti-NCAM1 antibody (catalog no. 563443, BD Biosciences), and AF647-conjugated anti-L1CAM antibody (catalog no. 564194, BD Biosciences) were used for detection of EV subpopulations.

Techniques: Marker, Single Particle, Gradient Centrifugation, Size-exclusion Chromatography, Transmission Assay, Labeling, Isolation, Clinical Proteomics, Staining, Derivative Assay

Journal: Science advances

Article Title: ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids.

doi: 10.1126/sciadv.adi3647

Figure Lengend Snippet: Fig. 2. Comparative analysis of NDEV candidate markers (ATP1A3, NCAM1, and L1CAM) expression in EVs by super-resolution microscopy. (A) Schematics showing the staining protocol of ATP1A3 and NCAM1 for EVs. (B) Representative super-resolution microscopy images showing the expression of ATP1A3 and NCAM1 in pan-tetraspanins (CD9/81/63) positive EVs at a single-particle level. Scale bars, 100 nm. (C) Pie charts showing the heterogeneous EV population with ATP1A3 and NCAM1 in different EV preparations. Particles with diameter less than 200 nm were quantified. (D) Schematics showing the staining protocol of ATP1A3 and L1CAM for EVs. (E) Representative super-resolution microscopy images showing the expression of ATP1A3 and L1CAM in pan-tetraspanins (CD9/81/63) positive EVs at a single- particle level. Scale bars, 100 nm. (F) Pie charts showing the heterogeneous EV population with ATP1A3 and L1CAM in different EV preparations. Particles with diameter less than 200 nm were quantified.

Article Snippet: In our experiment, EV antibodies cocktail (EV-TC-AB-01, NanoView Biosciences) containing AF488-conjugated anti-CD63, anti-CD81, and anti-CD9 antibodies, AF647-conjugated anti-ATP1A3 antibody (NBP299238AF647, Novus Biologicals), AF647-conjugated anti-NCAM1 antibody (catalog no. 563443, BD Biosciences), and AF647-conjugated anti-L1CAM antibody (catalog no. 564194, BD Biosciences) were used for detection of EV subpopulations.

Techniques: Expressing, Super-Resolution Microscopy, Staining, Single Particle

Journal: Science advances

Article Title: ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids.

doi: 10.1126/sciadv.adi3647

Figure Lengend Snippet: Fig. 3. ATP1A3+ EV immunoprecipitated from human brain tissues displays higher neuronal cell type specificity. (A) Workflow for characterizing ATP1A3+, NCAM1+, and L1CAM+ EVs immunoprecipitated from brain. (B) Western blot images of 5% total EVs (Input), supernatant (S.P), and immunoprecipitated EVs (A, ATP1A3; L, L1CAM; N, NCAM1) isolated from control brains (n = 3) and probed for neuron and glia markers. Equivalent proteins from brain-EVs were used for immuno- capturing ATP1A3, L1CAM, and NCAM1. IB, immunoblotting antibody (C) Principal components analysis (PCA). The EV proteome of total brain-EV and immunoprecip- itated EVs were measured in triplicate and classified. (D) Venn diagram showing the number of proteins differentially identified in total, ATP1A3+, L1CAM+, and NCAM1+

Article Snippet: In our experiment, EV antibodies cocktail (EV-TC-AB-01, NanoView Biosciences) containing AF488-conjugated anti-CD63, anti-CD81, and anti-CD9 antibodies, AF647-conjugated anti-ATP1A3 antibody (NBP299238AF647, Novus Biologicals), AF647-conjugated anti-NCAM1 antibody (catalog no. 563443, BD Biosciences), and AF647-conjugated anti-L1CAM antibody (catalog no. 564194, BD Biosciences) were used for detection of EV subpopulations.

Techniques: Immunoprecipitation, Western Blot, Isolation, Control

Journal: Science advances

Article Title: ATP1A3 as a target for isolating neuron-specific extracellular vesicles from human brain and biofluids.

doi: 10.1126/sciadv.adi3647

Figure Lengend Snippet: Fig. 4. ATP1A3+ EV analysis in human CSF and plasma. (A) Workflow for characterizing ATP1A3+ EVs immunoprecipitated from CSF. (B) Simoa quantification of AD related neuropathogenic proteins (Aβ42, Aβ40, T-tau, P-tau181, and SNAP25) in ATP1A3+ CSF-EVs from healthy controls (n = 3) and AD (n = 3). Data are presented by box and whisker plot which shows the median and the 25th to 75th percentiles. (C) Aβ42/Aβ40 ratio in CSF, total CSF-EV, and ATP1A3 immunoprecipitation (IP) CSF-EV samples among AD group. (D) Workflow explaining single-particle analysis of Aβ+ population in ATP1A3+ plasma-EV to distinguish AD from healthy controls (CTL) and mild cognitive impairment (MCI). (E) Representative super-resolution microscopy images showing plasma-derived Aβ+ ATP1A3+ single EV among control, MCI, and AD groups [green: FITC-CD9/81/63, red: Alexa Flour 555-ATP1A3, gray: Alexa Flour 647-Aβ (clone 4G8)]. Scale bars, 100 nm. (F) Percentage of Aβ+ population in ATP1A3+ plasma-EV among control, MCI, and AD groups (n = 10 per group). (G) Correlations of Aβ+ population in ATP1A3+ plasma-EV with clinical Braak stage, Mini- Mental State Examination (MMSE), and Dementia Rating Scale (DRS) scores in combined control (n = 7), MCI (n = 10), and AD (n = 10) samples. The dashed line indicates 95% confidence band of the best-fit line. Pearson correlation coefficients are shown. (H) Quantification of Aβ42/Aβ40 level in total plasma samples from CTL (n = 9), MCI (n = 10), and AD (n = 10). (I to J) Receiver-operating characteristics (ROC) curves displaying the performance of Aβ+ population in ATP1A3+ plasma-EV, plasma Aβ40, Aβ42, Aβ42/Aβ40 ratio, and T-tau to distinguish (I) AD from CTL and (J) AD from MCI. T-tau, total tau; AUC, area under the curve; CI, confidence interval; ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In our experiment, EV antibodies cocktail (EV-TC-AB-01, NanoView Biosciences) containing AF488-conjugated anti-CD63, anti-CD81, and anti-CD9 antibodies, AF647-conjugated anti-ATP1A3 antibody (NBP299238AF647, Novus Biologicals), AF647-conjugated anti-NCAM1 antibody (catalog no. 563443, BD Biosciences), and AF647-conjugated anti-L1CAM antibody (catalog no. 564194, BD Biosciences) were used for detection of EV subpopulations.

Techniques: Clinical Proteomics, Immunoprecipitation, Whisker Assay, Single Particle, Super-Resolution Microscopy, Derivative Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Microglial-derived miRNA let-7 and HMGB1 contribute to ethanol-induced neurotoxicity via TLR7

doi: 10.1186/s12974-017-0799-4

Figure Lengend Snippet: Ethanol causes microvesicle (MV) release of let-7b and HMGB1 from microglia. a MVs were isolated from hippocampal-entorrhinal slice culture (HEC) media after ethanol exposure. let-7b was increased in MVs 3.7-fold by ethanol. b Ethanol increased let-7b in BV2 microglia-derived MVs by 3-fold; 326.7 ± 76.3% vs 100 ± 18.2%, Ethanol vs Control, mean ± SEM, * p < 0.05, t- test. c SH-SY5Y neurons were treated with 100 mM ethanol for 24 h. Ethanol had no effect on let-7b release in MVs from SH-SY5Y neurons; 95.75 ± 5.2% vs 100 ± 8.1%, Control vs Ethanol, mean ± SEM. d Ethanol increased HMGB1 secretion into HEC culture media in a dose dependent fashion. e 72.9% of HMGB1+ MVs from HEC media are microglial-derived, whereas astrocytes and neurons comprised 15.3 and 11.8%, respectively. One-way ANOVA with Sidak’s multiple comparisons test, **** p < 0.00001. f Representative histogram of one ethanol treated sample showing populations of astrocytic (GFAP, dark gray ), microglial (CD11b, light blue ), and neuron-derived HMGB1+ MVs (Na/K ATPase α3, light gray ). * p < 0.05, ** p < 0.01, mean ± SEM, N = 3–6 per group

Article Snippet: HMGB1 ELISA kit was purchased from IBL International (Hamburg, Germany); primary antibodies from Novus Biologicals: Na + /K + ATPase α3 (NB300-540APC), GFAP (NBP2-34401 V2), CD11b (NBP2-34678PE), and HMGB1 (NB100-2322AF488); and primary antibodies from Abcam: CD11b for western blot (ab75476), argonaute 2 (ab32381).

Techniques: Isolation, Derivative Assay, 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 primary antibodies used were as follows: anti-α2 Na,K-ATPase (1:200, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-GLAST (1:600, mouse monoclonal; Novocastra Laboratories), anti-GLAST (1:200, rabbit polyclonal; Santa Cruz Biotechnology), anti-α3 Na,K-ATPase (1:200, clone XVIF9-G10; Novus Biologicals), anti-GLT-1a (1:500, rabbit polyclonal; Affinity BioReagents), anti-GLT-1b (1:200, mouse monoclonal, clone 10B7; Abcam), and anti-glial fibrillary acidic protein (GFAP) (1:1000, mouse monoclonal, clone GA5; Millipore Bioscience Research Reagents).

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 primary antibodies used were as follows: anti-α2 Na,K-ATPase (1:200, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-GLAST (1:600, mouse monoclonal; Novocastra Laboratories), anti-GLAST (1:200, rabbit polyclonal; Santa Cruz Biotechnology), anti-α3 Na,K-ATPase (1:200, clone XVIF9-G10; Novus Biologicals), anti-GLT-1a (1:500, rabbit polyclonal; Affinity BioReagents), anti-GLT-1b (1:200, mouse monoclonal, clone 10B7; Abcam), and anti-glial fibrillary acidic protein (GFAP) (1:1000, mouse monoclonal, clone GA5; Millipore Bioscience Research Reagents).

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 primary antibodies used were as follows: anti-α2 Na,K-ATPase (1:200, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-GLAST (1:600, mouse monoclonal; Novocastra Laboratories), anti-GLAST (1:200, rabbit polyclonal; Santa Cruz Biotechnology), anti-α3 Na,K-ATPase (1:200, clone XVIF9-G10; Novus Biologicals), anti-GLT-1a (1:500, rabbit polyclonal; Affinity BioReagents), anti-GLT-1b (1:200, mouse monoclonal, clone 10B7; Abcam), and anti-glial fibrillary acidic protein (GFAP) (1:1000, mouse monoclonal, clone GA5; Millipore Bioscience Research Reagents).

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 primary antibodies used were as follows: anti-α2 Na,K-ATPase (1:200, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-GLAST (1:600, mouse monoclonal; Novocastra Laboratories), anti-GLAST (1:200, rabbit polyclonal; Santa Cruz Biotechnology), anti-α3 Na,K-ATPase (1:200, clone XVIF9-G10; Novus Biologicals), anti-GLT-1a (1:500, rabbit polyclonal; Affinity BioReagents), anti-GLT-1b (1:200, mouse monoclonal, clone 10B7; Abcam), and anti-glial fibrillary acidic protein (GFAP) (1:1000, mouse monoclonal, clone GA5; Millipore Bioscience Research Reagents).

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 primary antibodies used were as follows: anti-α2 Na,K-ATPase (1:200, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-GLAST (1:600, mouse monoclonal; Novocastra Laboratories), anti-GLAST (1:200, rabbit polyclonal; Santa Cruz Biotechnology), anti-α3 Na,K-ATPase (1:200, clone XVIF9-G10; Novus Biologicals), anti-GLT-1a (1:500, rabbit polyclonal; Affinity BioReagents), anti-GLT-1b (1:200, mouse monoclonal, clone 10B7; Abcam), and anti-glial fibrillary acidic protein (GFAP) (1:1000, mouse monoclonal, clone GA5; Millipore Bioscience Research Reagents).

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 primary antibodies used were as follows: anti-α2 Na,K-ATPase (1:200, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-GLAST (1:600, mouse monoclonal; Novocastra Laboratories), anti-GLAST (1:200, rabbit polyclonal; Santa Cruz Biotechnology), anti-α3 Na,K-ATPase (1:200, clone XVIF9-G10; Novus Biologicals), anti-GLT-1a (1:500, rabbit polyclonal; Affinity BioReagents), anti-GLT-1b (1:200, mouse monoclonal, clone 10B7; Abcam), and anti-glial fibrillary acidic protein (GFAP) (1:1000, mouse monoclonal, clone GA5; Millipore Bioscience Research Reagents).

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 primary antibodies used were as follows: anti-α2 Na,K-ATPase (1:200, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-GLAST (1:600, mouse monoclonal; Novocastra Laboratories), anti-GLAST (1:200, rabbit polyclonal; Santa Cruz Biotechnology), anti-α3 Na,K-ATPase (1:200, clone XVIF9-G10; Novus Biologicals), anti-GLT-1a (1:500, rabbit polyclonal; Affinity BioReagents), anti-GLT-1b (1:200, mouse monoclonal, clone 10B7; Abcam), and anti-glial fibrillary acidic protein (GFAP) (1:1000, mouse monoclonal, clone GA5; Millipore Bioscience Research Reagents).

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 primary antibodies used were as follows: anti-α2 Na,K-ATPase (1:200, rabbit polyclonal; Millipore Bioscience Research Reagents), anti-GLAST (1:600, mouse monoclonal; Novocastra Laboratories), anti-GLAST (1:200, rabbit polyclonal; Santa Cruz Biotechnology), anti-α3 Na,K-ATPase (1:200, clone XVIF9-G10; Novus Biologicals), anti-GLT-1a (1:500, rabbit polyclonal; Affinity BioReagents), anti-GLT-1b (1:200, mouse monoclonal, clone 10B7; Abcam), and anti-glial fibrillary acidic protein (GFAP) (1:1000, mouse monoclonal, clone GA5; Millipore Bioscience Research Reagents).

Techniques: Functional Assay

Journal: Scientific Reports

Article Title: The Warburg Effect Mediator Pyruvate Kinase M2 Expression and Regulation in the Retina

doi: 10.1038/srep37727

Figure Lengend Snippet: Retinal homogenates from dark- and light-adapted Balb/c mice were subjected to OptiPrep™ (8–40%) density gradient centrifugation ( A ). Fractions of inner segments and intact photoreceptors ( B ) were collected from the top to the bottom of the gradients. A ten-microliter sample (one-microliter for rhodopsin) was subjected to immunoblot analysis ( C ) with opsin, α-3 Na/K ATPase, pPKM2, PKM2, and PKM1 antibodies. Full-length blots are presented in the .

Article Snippet: Rabbit polyclonal anti-red/green cone opsin (M-opsin) antibody was obtained from Millipore (Billerica, MA). α-3 Na/K ATPase antibody was obtained from Novus Biologicals (Littleton, CO).

Techniques: Gradient Centrifugation, Western Blot

Journal: Scientific Reports

Article Title: The Warburg Effect Mediator Pyruvate Kinase M2 Expression and Regulation in the Retina

doi: 10.1038/srep37727

Figure Lengend Snippet: Retinal homogenates from Nrl −/− mice were subjected to OptiPrep™ (8–40%) density gradient centrifugation, and fractions of inner segments and intact photoreceptors ( A ) were collected from the top to the bottom of the gradients. A ten-microliter sample (three-microliter for M-opsin) was subjected to immunoblot analysis ( B ) with M-opsin, α-3 Na/K ATPase, pPKM2, PKM2, and PKM1 antibodies. Full-length blots are presented in the .

Article Snippet: Rabbit polyclonal anti-red/green cone opsin (M-opsin) antibody was obtained from Millipore (Billerica, MA). α-3 Na/K ATPase antibody was obtained from Novus Biologicals (Littleton, CO).

Techniques: Gradient Centrifugation, Western Blot