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  • 99
    Thermo Fisher v atpase
    Effects of rotenone on synaptosomal [ 3 H]-DA uptake, and changes in protein levels of CoxIV, <t>V-ATPase</t> H and <t>SV2a</t> in 3-month-old mouse striatal synaptosomes. ( a ) Greater reduction in LRRK2 R1441G mutant synaptosomal [ 3 H]-DA uptake at 100 nM rotenone as compared to similarly treated WT ( WT: 7045 ± 583 cpm; mutant: 5311 ± 405 cpm ). Data represents mean ± standard error of mean (SEM) from at least seven independent experiments. (b) Protein expression levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. CoxIV and V-ATPase H levels in total striatal synaptosomal lysates were significantly lower in mutant mice compared to WT controls (N = 6). Statistical significance between groups was analyzed using Student’s unpaired t-test, *p
    V Atpase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore atpase antibody
    Decreased K ATP channel subunit expression in ankyrin-B +/− hearts. Detergent-soluble ankyrin-B +/− heart lysates expressed significantly less ankyrin-B ( A ), Kir6.2 ( B ), SUR1 ( C ), SUR2A ( D ), <t>Na/K-ATPase</t> ( H ), and NCX1 ( I ). There were no significant differences in the expression of ankyrin-G ( E ), Kir6.1 ( F ), or NHERF1 (loading control; G ). J , quantitative data demonstrating a significant decrease in the expression of ankyrin-B, NCX1, Na/K-ATPase, and K ATP channel proteins in ankyrin-B +/− compared with wild-type detergent-soluble mouse heart lysates. Expression of proteins was normalized to wild-type expression. IB , immunoblot. *, p
    Atpase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agrisera vacuolor adenosine triphosphatase v atpase antibody
    Decreased K ATP channel subunit expression in ankyrin-B +/− hearts. Detergent-soluble ankyrin-B +/− heart lysates expressed significantly less ankyrin-B ( A ), Kir6.2 ( B ), SUR1 ( C ), SUR2A ( D ), <t>Na/K-ATPase</t> ( H ), and NCX1 ( I ). There were no significant differences in the expression of ankyrin-G ( E ), Kir6.1 ( F ), or NHERF1 (loading control; G ). J , quantitative data demonstrating a significant decrease in the expression of ankyrin-B, NCX1, Na/K-ATPase, and K ATP channel proteins in ankyrin-B +/− compared with wild-type detergent-soluble mouse heart lysates. Expression of proteins was normalized to wild-type expression. IB , immunoblot. *, p
    Vacuolor Adenosine Triphosphatase V Atpase Antibody, supplied by Agrisera, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Biomol GmbH atpase assay adenosine triphosphatase atpase activity
    Decreased K ATP channel subunit expression in ankyrin-B +/− hearts. Detergent-soluble ankyrin-B +/− heart lysates expressed significantly less ankyrin-B ( A ), Kir6.2 ( B ), SUR1 ( C ), SUR2A ( D ), <t>Na/K-ATPase</t> ( H ), and NCX1 ( I ). There were no significant differences in the expression of ankyrin-G ( E ), Kir6.1 ( F ), or NHERF1 (loading control; G ). J , quantitative data demonstrating a significant decrease in the expression of ankyrin-B, NCX1, Na/K-ATPase, and K ATP channel proteins in ankyrin-B +/− compared with wild-type detergent-soluble mouse heart lysates. Expression of proteins was normalized to wild-type expression. IB , immunoblot. *, p
    Atpase Assay Adenosine Triphosphatase Atpase Activity, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc na k atpase
    Effect of garlic and its metabolites on Na + /K + <t>-ATPase</t> expression. (A,B) Western blot and bar graph of Na + /K + -ATPase from H9C2 cells. (C,D) Western blot and bar graph of Na + /K + -ATPase from rat heart. (Gels and blots were cropped and run under same experimental conditions). ( N = 3). Data were shown as mean ± SEM, ∗ p
    Na K Atpase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Cytoskeleton Inc atpases
    Effect of garlic and its metabolites on Na + /K + <t>-ATPase</t> expression. (A,B) Western blot and bar graph of Na + /K + -ATPase from H9C2 cells. (C,D) Western blot and bar graph of Na + /K + -ATPase from rat heart. (Gels and blots were cropped and run under same experimental conditions). ( N = 3). Data were shown as mean ± SEM, ∗ p
    Atpases, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    SAS institute atpases
    Effect of garlic and its metabolites on Na + /K + <t>-ATPase</t> expression. (A,B) Western blot and bar graph of Na + /K + -ATPase from H9C2 cells. (C,D) Western blot and bar graph of Na + /K + -ATPase from rat heart. (Gels and blots were cropped and run under same experimental conditions). ( N = 3). Data were shown as mean ± SEM, ∗ p
    Atpases, supplied by SAS institute, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    rdi research diagnostics atpase
    Cell fractionation of shortened MBP-GFP hybrid protein. Cells expressing MBP[SS128]-GFP were fractionated into inner and outer membranes as described in Materials and Methods. The immunoblot shown was decorated with anti-GFP antibody. MBP[SS128]-GFP migrated at a predicted molecular mass of 40 kDa and fractionated with the outer membrane. An apparent smaller-molecular-weight derivative of this protein was also found in the inner membrane fraction. Molecular weight (MW) standards are as shown. IM, inner membrane fraction; IM-W, KCl wash of inner membrane fraction; OM, outer membrane fraction; OM-W, KCl wash of outer membrane fraction. Although not shown, <t>OmpA</t> and <t>ATPase</t> were used to monitor fractionation of the outer membrane and inner membranes, respectively.
    Atpase, supplied by rdi research diagnostics, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher atpase
    <t>SDH,</t> mABC1, PIC, ANT, and <t>ATPase</t> interact with each other in co-IP studies on rat liver mitochondrial IM. IM of mitochondria was used as a control. ( A ) Co-IP of the 30-kDa component of SDH with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. ( B ) Co-IP of the 70-kDa components of SDH with Abs against 30- and 70-kDa of SDH, mABC1, ANT, ATPase, and PIC. ( C ) Co-IP of the α-subunit of ATPase with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. The α-subunit of ATPase has a molecular mass of ≈60 kDa. An Ab against the β-subunit of ATPase coimmunoprecipitated the same proteins. ( D ) Co-IP of PIC with Abs against 30- and 70-kDa component of SDH, ATPase, PIC, and mABC1. PIC has a molecular mass of ≈30 kDa. ( E ) Co-IP of mABC1 with Abs against 70-kDa components of SDH, ATPase, mABC1, and PIC. mABC1 has a molecular mass of ≈55 kDa. Negative controls included Abs against complex I and IV of the respiratory pathway, Kir6.1, and cyclophilin D. The IM of mitochondria was prepared in 1.2% digitonin and 6% lubrol WX and was dissolved in 300 mM KPi/10% ethylene glycol/5 mM EDTA/4 mM ATP/0.5 mM DTT, pH 7.9. The columns were also washed with 2–3% (octylphenoxy)polyethoxyethanol to increase the specificity of the interactions between proteins.
    Atpase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Upstate Biotechnology Inc k atpase
    Pancreatic juice contains CD39 in the particulate fraction A , pancreatic juice collected from a pancreas stimulated with secretin and later with CCK-8 was analysed for CD39 with Western blot. Samples for analysis were collected 15–45 min after stimulation. Lanes 2 and 3 show the untreated total juice; lanes 5 and 6 show the ultracentrifuged pellet and lanes 7 and 8 show the supernatant. Agonists are indicated in each lane. In addition, lanes 1 and 4 show the Western blot of whole pancreas tissue. B , Western blot with an antibody against the <t>α</t> subunit of the Na + ,K + <t>-ATPase</t> was performed on total juice from secretin and CCK-8 stimulated pancreas (lanes 10 and 11) and as a positive control on the whole pancreas (lane 9). The gel is the representative of at least 3 different experiments.
    K Atpase, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 90/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    MitoSciences mitochondrial atpase
    Pancreatic juice contains CD39 in the particulate fraction A , pancreatic juice collected from a pancreas stimulated with secretin and later with CCK-8 was analysed for CD39 with Western blot. Samples for analysis were collected 15–45 min after stimulation. Lanes 2 and 3 show the untreated total juice; lanes 5 and 6 show the ultracentrifuged pellet and lanes 7 and 8 show the supernatant. Agonists are indicated in each lane. In addition, lanes 1 and 4 show the Western blot of whole pancreas tissue. B , Western blot with an antibody against the <t>α</t> subunit of the Na + ,K + <t>-ATPase</t> was performed on total juice from secretin and CCK-8 stimulated pancreas (lanes 10 and 11) and as a positive control on the whole pancreas (lane 9). The gel is the representative of at least 3 different experiments.
    Mitochondrial Atpase, supplied by MitoSciences, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Trinity Biotech p97 atpase
    Pancreatic juice contains CD39 in the particulate fraction A , pancreatic juice collected from a pancreas stimulated with secretin and later with CCK-8 was analysed for CD39 with Western blot. Samples for analysis were collected 15–45 min after stimulation. Lanes 2 and 3 show the untreated total juice; lanes 5 and 6 show the ultracentrifuged pellet and lanes 7 and 8 show the supernatant. Agonists are indicated in each lane. In addition, lanes 1 and 4 show the Western blot of whole pancreas tissue. B , Western blot with an antibody against the <t>α</t> subunit of the Na + ,K + <t>-ATPase</t> was performed on total juice from secretin and CCK-8 stimulated pancreas (lanes 10 and 11) and as a positive control on the whole pancreas (lane 9). The gel is the representative of at least 3 different experiments.
    P97 Atpase, supplied by Trinity Biotech, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology v atpase
    Quantitative analyses of the expression levels of <t>CD147</t> and <t>V-ATPase</t> in breast cancer samples. Comparison of the expression levels of (A) CD147 and (B) V-ATPase between the chemotherapy-sensitive and -resistant groups. (C) Correlation between the H-scores of CD147 and V-ATPase in breast invasive ductal cancer. ***P
    V Atpase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Seca Inc cytoplasmic atpase
    Quantitative analyses of the expression levels of <t>CD147</t> and <t>V-ATPase</t> in breast cancer samples. Comparison of the expression levels of (A) CD147 and (B) V-ATPase between the chemotherapy-sensitive and -resistant groups. (C) Correlation between the H-scores of CD147 and V-ATPase in breast invasive ductal cancer. ***P
    Cytoplasmic Atpase, supplied by Seca Inc, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology atpase alpha3
    Quantitative analyses of the expression levels of <t>CD147</t> and <t>V-ATPase</t> in breast cancer samples. Comparison of the expression levels of (A) CD147 and (B) V-ATPase between the chemotherapy-sensitive and -resistant groups. (C) Correlation between the H-scores of CD147 and V-ATPase in breast invasive ductal cancer. ***P
    Atpase Alpha3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Epitomics k atpase
    Western blot of whole cell (WC) homogenates and apical membrane fractions from two immortalized hRPTC lines grown in Transwell membranes. Arrows on the left-hand side of the blots indicate the molecular sizes, while the arrows on the right-hand side indicate the molecular sizes of the bands of the proteins of interest. The blot was probed for <t>NBCe2</t> along with the following membrane markers: CD-13 (APN microvilli marker), Na + ,K + <t>/ATPase</t> (basolateral membrane marker), and NBCe1 (basolateral membrane marker). The results demonstrate that two isoforms of NBCe2 exist in the apical membrane of polarized RPTCs grown on Transwells. The control western blots demonstrate that αNa + ,K + /ATPase (NKA) and NBCe1 only stain weakly in the apical fraction, probably due to some basolateral contamination inherent in this kind of preparation. WC homogenates demonstrate the presence of NBCe2, NBCe1, CD-13, and Na + ,K + /ATPase, as expected.
    K Atpase, supplied by Epitomics, used in various techniques. Bioz Stars score: 98/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore k atpase
    Effect of vanadate on renal <t>α</t> 1 -Na, <t>K-ATPase</t> protein abundance. Ten days of vanadate injection significantly suppressed α 1 -Na, K-ATPase protein abundance. Histogram bars show the densitometric analyses ratios of α 1 -Na, K-ATPase to β-actin intensity, and the representative immunoblot photographs are present. All values are expressed as the mean ± SD of 8 rats/group. *Significantly different from the sham group ( p
    K Atpase, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Horizon Discovery v atpase
    Intracellular localization of subunit <t>a3</t> and markers for the Golgi apparatus and lysosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to the Golgi and lysosomes, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the <t>V-ATPase</t> and Giantin or LAMP2 as markers for the Golgi and lysosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the Golgi apparatus marker Giantin ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the lysosomal marker LAMP2 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.
    V Atpase, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher k atpase
    STED microscopy dissecting the localization of Na + ,K + <t>-ATPase</t> in cultured striatal neurons . The 5 × 3 mosaic shows a comparision of the confocal (left) and STED images (middle) of the Alexa-594 immunolabelled <t>α3</t> NKA in dendritic spines. Postprocessing of the raw STED data by a Richardson-Lucy deconvolution further enhances the details as shown in the right row of images. Scale bar: 500 nm
    K Atpase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Synaptic Systems v atpase
    STED microscopy dissecting the localization of Na + ,K + <t>-ATPase</t> in cultured striatal neurons . The 5 × 3 mosaic shows a comparision of the confocal (left) and STED images (middle) of the Alexa-594 immunolabelled <t>α3</t> NKA in dendritic spines. Postprocessing of the raw STED data by a Richardson-Lucy deconvolution further enhances the details as shown in the right row of images. Scale bar: 500 nm
    V Atpase, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 87/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore atpase assays
    Immunostaining of MCF10a cells overexpressing <t>subunit</t> a isoforms using an antibody against <t>V-ATPase.</t> MCF10a cells were grown as a monolayer on coverslips in 6-well plates. Cells were immunostained using an antibody against subunit A of V-ATPase. Images
    Atpase Assays, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam k atpase
    Profiling of membrane-fraction proteins from the prefrontal cortex of control and alcoholic subjects. Control (C) or alcoholic (A) prefrontal cortex membrane fraction proteins were resolved by 1D PAGE and proteins stained with colloidal Coomassie. Alcoholic subjects displayed a prominent reduction in the level of protein staining at a protein band of ∼270 <t>kDa</t> and upregulation at ∼112 kDa (upper panel). Protein levels of transmembranous (∼112 kDa) Na + ,K + <t>-ATPase</t> α-subunit, and oligomeric forms of the α-subunit were visualized by Western blotting (middle panel). The level of transmembranous α-subunit was quantified using actin for normalisation (lower panel). For marked significance: *** = p
    K Atpase, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Accurate Chemical & Scientific Corporation k atpase
    Electron microscopical analysis of caveolin-1 in cultured human ARPE-19 cells. (A) Micrograph of an ARPE-19 cell depicting apical microvilli (AM) and basal infoldings (BI). The nucleus (N) can also be seen (7700×). Panel A serves as a comparison for panels B–E, which are higher magnifications of the apical and basal plasma membranes of a RPE cell. (B) Higher magnification of apical microvilli labelled with an antibody against caveolin-1. No gold particles were observed (60,000×). (C) Higher magnification of the basal infoldings of RPE labelled with an antibody against caveolin-1. Arrows point to gold particles (35,500×). (D) Higher magnification of apical microvilli labelled with antibody against the Na + ,K + <t>-ATPase.</t> Gold particles were abundant (46,000×). (E) Higher magnification of the basolateral infoldings of A RPE cells labelled with an antibody against folate receptor <t>α</t> . Gold particles were present across the membrane (46,000×).
    K Atpase, supplied by Accurate Chemical & Scientific Corporation, used in various techniques. Bioz Stars score: 90/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc k atpase
    Identification of galactose as a hypoglycemic compound using the hyperglycemic silkworm model. (A) Preparation protocol for the jiou extract. (B) Silkworms were fed a 10% (w/w) glucose diet for 60 min. 50 µl of Jiou extract (1 mg/ml) or human insulin (3.5 mg/ml) was injected into the hemolymph of the hyperglycemic silkworms. The silkworms were fasted for 6 h and the sugar level in the hemolymph was determined. n = 6–7 per group. Data are shown as means ± SD. (C, D) The jiou extract was treated with TFA, and analyzed by TLC. Sugars were localized with 10% sulfuric acid solution. (E) Silkworms were fed a 10% (w/w) glucose diet for 60 min. 50 µl of D-Galactose (Gal), D-glucose (Glu), D-talose (Tal), D-mannose (Man), (1 mg/ml) or human insulin (3.5 mg/ml) was injected into the hemolymph of hyperglycemic silkworms. Silkworms were fasted for 6 h and sugar level in the hemolymph was determined. n = 6 per group. (F) Structure of sugar is shown by Fischer projection in the panel. Numbers shown on the left indicate the carbon positions of the sugar. Red hydroxyl group indicates the positions that differ from D-galactose. Activity represents the hypoglycemic effect. Data are shown as means ± SD. NS; not significant. (G) Galactose (10 mg/ml, 0.5 ml i.p.) was injected to streptozotocin induced-diabetic mice, and blood glucose level was determined after 4 h of fasting. Blood glucose levels in streptozotocin-induced hyperglycemic mice were measured (blood glucose 250–400 mg/dl) and then the mice were treated with either PBS or galactose solution. Four hours after administration and the removal of diet, the blood glucose levels were measured again. The data represent the blood glucose value after treatment divided by the blood glucose value before treatment of individual animals. In all panels, the statistical significance of the difference was evaluated using Student's t test. (H) Blood glucose levels in streptozotocin-induced hyperglycemic mice were measured (blood glucose 250–400 mg/dl) and then the mice were treated with either PBS, galactose (200 mg/kg mouse, i.p.), or glucose (200 mg/kg mouse, i.p.) solution. Two hours after administration and removal of the diet, the mice were killed and the membrane fraction in mouse liver was isolated. <t>GLUT2</t> and Na, <t>K-ATPase</t> were detected by Western blot analysis with anti-GLUT2 antibody or anti-Na, K-ATPase antibody. Immunoblots of GLUT2 and Na, K-ATPase (Top) and calculations of relative GLUT2 (Bottom). n = 3–4 per group. Data at the bottom of the figure are shown as means ± SD. In all panels, the statistical significance of the difference was evaluated using Student's t test.
    K Atpase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank k atpase
    The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + <t>-ATPase</t> <t>(NKA),</t> and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P
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    The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + <t>-ATPase</t> <t>(NKA),</t> and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P
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    Tocris atpase inhibitor
    The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + <t>-ATPase</t> <t>(NKA),</t> and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P
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    Seca Inc atpase seca
    The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + <t>-ATPase</t> <t>(NKA),</t> and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P
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    Millipore control atpase
    The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + <t>-ATPase</t> <t>(NKA),</t> and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P
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    MBL International k atpase
    The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + <t>-ATPase</t> <t>(NKA),</t> and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P
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    Becton Dickinson mitochondrial atpase
    The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + <t>-ATPase</t> <t>(NKA),</t> and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P
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    Image Search Results


    Effects of rotenone on synaptosomal [ 3 H]-DA uptake, and changes in protein levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. ( a ) Greater reduction in LRRK2 R1441G mutant synaptosomal [ 3 H]-DA uptake at 100 nM rotenone as compared to similarly treated WT ( WT: 7045 ± 583 cpm; mutant: 5311 ± 405 cpm ). Data represents mean ± standard error of mean (SEM) from at least seven independent experiments. (b) Protein expression levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. CoxIV and V-ATPase H levels in total striatal synaptosomal lysates were significantly lower in mutant mice compared to WT controls (N = 6). Statistical significance between groups was analyzed using Student’s unpaired t-test, *p

    Journal: Scientific Reports

    Article Title: Combined LRRK2 mutation, aging and chronic low dose oral rotenone as a model of Parkinson’s disease

    doi: 10.1038/srep40887

    Figure Lengend Snippet: Effects of rotenone on synaptosomal [ 3 H]-DA uptake, and changes in protein levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. ( a ) Greater reduction in LRRK2 R1441G mutant synaptosomal [ 3 H]-DA uptake at 100 nM rotenone as compared to similarly treated WT ( WT: 7045 ± 583 cpm; mutant: 5311 ± 405 cpm ). Data represents mean ± standard error of mean (SEM) from at least seven independent experiments. (b) Protein expression levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. CoxIV and V-ATPase H levels in total striatal synaptosomal lysates were significantly lower in mutant mice compared to WT controls (N = 6). Statistical significance between groups was analyzed using Student’s unpaired t-test, *p

    Article Snippet: COX IV (1:2000, Abcam #ab16056, 16 kD), NDUSF4 (1:500, Santa-Cruz #sc-100567, 18 kD), TH (1:2000, Millipore #MAB318, 58 kD), Actin (1:500, Santa-Cruz #sc-1615, 43 kD), Synaptophysin (1:2000, Cell Signaling #D35E4, 38 kD), V-ATPase (1:1000, ThermoFisher #PA5-22134, 55 kD), SV2a (1:1000, Santa-Cruz #sc-11936, 93 kD) were the primary antibodies used.

    Techniques: Mutagenesis, Expressing, Mouse Assay

    Decreased K ATP channel subunit expression in ankyrin-B +/− hearts. Detergent-soluble ankyrin-B +/− heart lysates expressed significantly less ankyrin-B ( A ), Kir6.2 ( B ), SUR1 ( C ), SUR2A ( D ), Na/K-ATPase ( H ), and NCX1 ( I ). There were no significant differences in the expression of ankyrin-G ( E ), Kir6.1 ( F ), or NHERF1 (loading control; G ). J , quantitative data demonstrating a significant decrease in the expression of ankyrin-B, NCX1, Na/K-ATPase, and K ATP channel proteins in ankyrin-B +/− compared with wild-type detergent-soluble mouse heart lysates. Expression of proteins was normalized to wild-type expression. IB , immunoblot. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart *

    doi: 10.1074/jbc.M110.147868

    Figure Lengend Snippet: Decreased K ATP channel subunit expression in ankyrin-B +/− hearts. Detergent-soluble ankyrin-B +/− heart lysates expressed significantly less ankyrin-B ( A ), Kir6.2 ( B ), SUR1 ( C ), SUR2A ( D ), Na/K-ATPase ( H ), and NCX1 ( I ). There were no significant differences in the expression of ankyrin-G ( E ), Kir6.1 ( F ), or NHERF1 (loading control; G ). J , quantitative data demonstrating a significant decrease in the expression of ankyrin-B, NCX1, Na/K-ATPase, and K ATP channel proteins in ankyrin-B +/− compared with wild-type detergent-soluble mouse heart lysates. Expression of proteins was normalized to wild-type expression. IB , immunoblot. *, p

    Article Snippet: Protein A-conjugated agarose beads (Rockland Immunochemicals, Inc.) were incubated with either control IgG or affinity-purified anti-ankyrin-B, anti-Kir6.2, anti-NCX1, or anti-Na/K-ATPase antibody in co-immunoprecipitation binding buffer (PBS with 0.1% Triton X-100 and protease inhibitor mixture (Sigma)) for 12 h at 4 °C.

    Techniques: Expressing

    Ankyrin-B forms a macromolecular complex with K ATP , Na/K-ATPase, and NCX that is reduced in ankyrin-B +/− heart. A–D , detergent-soluble lysates from wild-type and ankyrin-B +/− mouse hearts were used for co-immunoprecipitations with the indicated antibodies. IB , immunoblot; IP , immunoprecipitation. Co-immunoprecipitations of ankyrin-B +/− lysates used doubled amounts of input lysate to compensate for the reduction of ankyrin-B. E and F , detergent-soluble lysates from wild-type and ankyrin-B +/− mouse hearts were used for control co-immunoprecipitations, demonstrating no interaction of Na/K-ATPase ( NKA ) and NCX with SERCA2, Ca v 1.2, and Na v 1.5.

    Journal: The Journal of Biological Chemistry

    Article Title: Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart *

    doi: 10.1074/jbc.M110.147868

    Figure Lengend Snippet: Ankyrin-B forms a macromolecular complex with K ATP , Na/K-ATPase, and NCX that is reduced in ankyrin-B +/− heart. A–D , detergent-soluble lysates from wild-type and ankyrin-B +/− mouse hearts were used for co-immunoprecipitations with the indicated antibodies. IB , immunoblot; IP , immunoprecipitation. Co-immunoprecipitations of ankyrin-B +/− lysates used doubled amounts of input lysate to compensate for the reduction of ankyrin-B. E and F , detergent-soluble lysates from wild-type and ankyrin-B +/− mouse hearts were used for control co-immunoprecipitations, demonstrating no interaction of Na/K-ATPase ( NKA ) and NCX with SERCA2, Ca v 1.2, and Na v 1.5.

    Article Snippet: Protein A-conjugated agarose beads (Rockland Immunochemicals, Inc.) were incubated with either control IgG or affinity-purified anti-ankyrin-B, anti-Kir6.2, anti-NCX1, or anti-Na/K-ATPase antibody in co-immunoprecipitation binding buffer (PBS with 0.1% Triton X-100 and protease inhibitor mixture (Sigma)) for 12 h at 4 °C.

    Techniques: Immunoprecipitation

    TRPM2 channels are expressed in the sarcolemma and transverse tubules of adult cardiac myocytes. Confocal images of adult WT mouse ventricular myocytes labeled with anti-α 1 -subunit of Na + -K + -ATPase antibody ( A and D ), with ( B ) or without ( E ) rabbit

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury

    doi: 10.1152/ajpheart.00906.2012

    Figure Lengend Snippet: TRPM2 channels are expressed in the sarcolemma and transverse tubules of adult cardiac myocytes. Confocal images of adult WT mouse ventricular myocytes labeled with anti-α 1 -subunit of Na + -K + -ATPase antibody ( A and D ), with ( B ) or without ( E ) rabbit

    Article Snippet: Immunolocalization experiments indicated that both TRPM2 channels and the α1 -subunit of Na+ -K+ -ATPase (sarcolemma marker) were expressed in the sarcolemma and transverse tubules in adult LV myocytes ( ).

    Techniques: Labeling

    I/R decreases Na + -K + -ATPase current ( I pump ) more in TRPM2 KO myocytes. WT or KO hearts were subjected to either sham operation or 30 min ischemia of followed by 3 days of reperfusion before myocyte isolation. LV myocytes were held at 0 mV and 30°C.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury

    doi: 10.1152/ajpheart.00906.2012

    Figure Lengend Snippet: I/R decreases Na + -K + -ATPase current ( I pump ) more in TRPM2 KO myocytes. WT or KO hearts were subjected to either sham operation or 30 min ischemia of followed by 3 days of reperfusion before myocyte isolation. LV myocytes were held at 0 mV and 30°C.

    Article Snippet: Immunolocalization experiments indicated that both TRPM2 channels and the α1 -subunit of Na+ -K+ -ATPase (sarcolemma marker) were expressed in the sarcolemma and transverse tubules in adult LV myocytes ( ).

    Techniques: Isolation

    a) Effects of digitoxin on the level of ATF3, EGR1 protein. MDA-MB-453 cells were treated with 0, 0.1, 0.2 or 1 μ g/ml of digitoxin and after 1 and 24 h extracts were prepared and analyzed by Western blotting; an antibody to β-actin was used as a loading control. b) siRNA to ERK2. Cell were pretreated with control (nonsilencing) or MAPK1 (ERK2) siRNA for 24 h, exposed to digitoxin (0.4 μg/ml) for 24 h and percent inhibition of cell proliferation determined by the MTT assay. Percentages are normalized to DMSO. c) Inhibition of Na+-K+-ATPase activity in response to increasing concentrations of paclitaxel or digitoxin. The Na+-K+-ATPase assay was performed as described in Materials and Methods. The DMSO controls contained 3.3% DMSO. BARS: SD of triplicate assays.

    Journal: Journal of Carcinogenesis

    Article Title: Digitoxin activates EGR1 and synergizes with paclitaxel on human breast cancer cells

    doi: 10.4103/1477-3163.72578

    Figure Lengend Snippet: a) Effects of digitoxin on the level of ATF3, EGR1 protein. MDA-MB-453 cells were treated with 0, 0.1, 0.2 or 1 μ g/ml of digitoxin and after 1 and 24 h extracts were prepared and analyzed by Western blotting; an antibody to β-actin was used as a loading control. b) siRNA to ERK2. Cell were pretreated with control (nonsilencing) or MAPK1 (ERK2) siRNA for 24 h, exposed to digitoxin (0.4 μg/ml) for 24 h and percent inhibition of cell proliferation determined by the MTT assay. Percentages are normalized to DMSO. c) Inhibition of Na+-K+-ATPase activity in response to increasing concentrations of paclitaxel or digitoxin. The Na+-K+-ATPase assay was performed as described in Materials and Methods. The DMSO controls contained 3.3% DMSO. BARS: SD of triplicate assays.

    Article Snippet: Digitoxin or paclitaxel was added to ATPase (0.05 ml, 0.5 unit/ml, Sigma-Aldrich, St. Louis, MO, USA), mixed and equilibrated for 5 min at 37°C.

    Techniques: Multiple Displacement Amplification, Western Blot, Inhibition, MTT Assay, Activity Assay, ATPase Assay

    Effect of garlic and its metabolites on Na + /K + -ATPase expression. (A,B) Western blot and bar graph of Na + /K + -ATPase from H9C2 cells. (C,D) Western blot and bar graph of Na + /K + -ATPase from rat heart. (Gels and blots were cropped and run under same experimental conditions). ( N = 3). Data were shown as mean ± SEM, ∗ p

    Journal: Frontiers in Pharmacology

    Article Title: Novel Sulfur Metabolites of Garlic Attenuate Cardiac Hypertrophy and Remodeling through Induction of Na+/K+-ATPase Expression

    doi: 10.3389/fphar.2017.00018

    Figure Lengend Snippet: Effect of garlic and its metabolites on Na + /K + -ATPase expression. (A,B) Western blot and bar graph of Na + /K + -ATPase from H9C2 cells. (C,D) Western blot and bar graph of Na + /K + -ATPase from rat heart. (Gels and blots were cropped and run under same experimental conditions). ( N = 3). Data were shown as mean ± SEM, ∗ p

    Article Snippet: Garlic and Its Metabolites Failed to Show Any Anti-Hypertrophic Effect in H9C2 Cells in Presence of Na+ /K+ -ATPase Inhibitor To confirm whether garlic and its metabolites showed their anti-hypertrophic effect through Na+ /K+ -ATPase, we treated H9C2 cells with Iso in presence and absence of digoxin, a Na+ /K+ -ATPase inhibitor.

    Techniques: Expressing, Western Blot

    Membrane and cytoskeletal association of TRPC3, TRPC6, and TRPC3/TRPC6 chimeras. Proteins from transfected HEK 293T cells ( A–C ) or UT-7/Epo cells ( C ) were fractionated, purified, and analyzed by Western blotting ( WB ). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Quality of fractionation was confirmed by probing Western blots with anti-Na + K + -ATPase (membrane), anti-vimentin (cytoskeletal fraction), and anti-GAPDH (cytosol marker) antibodies. Equivalent input from lysates was confirmed by probing with anti-actin. A , fractionation with the Qiagen cell fractionation kit. Membrane and cytoskeletal association of TRPC3/6 channels and chimeras. Three to nine experiments (depending on the FLAG-tagged construct) were performed, and representative results are shown. B , fractionation using the 0.5% Triton X-100 extraction method. Representative results of three experiments are shown. C , subcellular fractionation of endogenous TRPC3 and TRPC6 in UT-7/Epo cells with the Qiagen fractionation kit. For all preparations, 100 μg/lane was loaded except for the cytoskeletal fraction of transfected cells, where 50 μg was loaded per lane. Endogenous TRPC3 was detected using anti-TRPC3-C antibody. Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody. Transfected HEK 293T cells were used as controls. Representative results of four experiments are shown. lys , whole cell lysate; M , membrane fraction; Csk , cytoskeletal fraction. *, bands that did not disappear with peptide blocking, indicating that they are nonspecific.

    Journal: The Journal of Biological Chemistry

    Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

    doi: 10.1074/jbc.M111.238360

    Figure Lengend Snippet: Membrane and cytoskeletal association of TRPC3, TRPC6, and TRPC3/TRPC6 chimeras. Proteins from transfected HEK 293T cells ( A–C ) or UT-7/Epo cells ( C ) were fractionated, purified, and analyzed by Western blotting ( WB ). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Quality of fractionation was confirmed by probing Western blots with anti-Na + K + -ATPase (membrane), anti-vimentin (cytoskeletal fraction), and anti-GAPDH (cytosol marker) antibodies. Equivalent input from lysates was confirmed by probing with anti-actin. A , fractionation with the Qiagen cell fractionation kit. Membrane and cytoskeletal association of TRPC3/6 channels and chimeras. Three to nine experiments (depending on the FLAG-tagged construct) were performed, and representative results are shown. B , fractionation using the 0.5% Triton X-100 extraction method. Representative results of three experiments are shown. C , subcellular fractionation of endogenous TRPC3 and TRPC6 in UT-7/Epo cells with the Qiagen fractionation kit. For all preparations, 100 μg/lane was loaded except for the cytoskeletal fraction of transfected cells, where 50 μg was loaded per lane. Endogenous TRPC3 was detected using anti-TRPC3-C antibody. Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody. Transfected HEK 293T cells were used as controls. Representative results of four experiments are shown. lys , whole cell lysate; M , membrane fraction; Csk , cytoskeletal fraction. *, bands that did not disappear with peptide blocking, indicating that they are nonspecific.

    Article Snippet: Blots were incubated with anti-TRPC3 (1:400; Alomone Laboratories, Jerusalem, Israel), anti-TRPC3-C targeted to the human C-terminal sequence RRRRLQDIEMGMGNSKSRLN (1:500 to 1:1000, Bethyl Laboratories, Inc., Montgomery, TX) , anti-TRPC3-N targeted to the murine N-terminal sequence LNGDLESAEPLERHGHKASL (1:1000; Bethyl Laboratories, Inc.) , anti-TRPC6 (1:250; Alomone Laboratories), anti-V5-HRP (1:10,000; Invitrogen), anti-FLAG (1:1000; Sigma), anti-PLCγ (SC-81, 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Epo-R (SC-697; 1:1000; Santa Cruz Biotechnology, Inc.), anti-actin (1:10,000; Sigma), anti-tubulin (1:10,000; Sigma), anti-GAPDH (1:5000; Cell Signaling Technology, Inc., Boston, MA), anti-Na+ K+ -ATPase (1:1000; Cell Signaling Technology, Inc.), anti-lamin (1:1000; Cell Signaling Technology, Inc.), anti-vimentin (1:1000; Cell Signaling Technology), or streptavidin-HRP (Pierce) antibodies.

    Techniques: Transfection, Purification, Western Blot, Construct, Fractionation, Marker, Cell Fractionation, Blocking Assay

    Subcellular localization of TRPC3, TRPC6, TRPC3/TRPC6 chimeras, PLCγ, and Epo-R. A , proteins from HEK 293T cells transfected with FLAG-TRPC3, FLAG-TRPC3-C6TRP, FLAG-TRPC6, FLAG-TRPC6-C3TRP-C3C2, and Epo-R were fractionated, purified, and analyzed by Western blotting ( WB ). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Transfected Epo-R was detected with anti-Epo-R antibody, and endogenous PLCγ was detected with anti-PLCγ antibody. Results from four fractionation experiments using FLAG-tagged constructs and two experiments using V5-tagged constructs were similar, and representative results with FLAG constructs are shown. B , representative results showing quality of fractionation by probing Western blots with anti-GAPDH, anti-Na + K + -ATPase, anti-lamin, and anti-vimentin (markers for cytosol, membrane, nuclear, and cytoskeletal fractions, respectively).

    Journal: The Journal of Biological Chemistry

    Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

    doi: 10.1074/jbc.M111.238360

    Figure Lengend Snippet: Subcellular localization of TRPC3, TRPC6, TRPC3/TRPC6 chimeras, PLCγ, and Epo-R. A , proteins from HEK 293T cells transfected with FLAG-TRPC3, FLAG-TRPC3-C6TRP, FLAG-TRPC6, FLAG-TRPC6-C3TRP-C3C2, and Epo-R were fractionated, purified, and analyzed by Western blotting ( WB ). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Transfected Epo-R was detected with anti-Epo-R antibody, and endogenous PLCγ was detected with anti-PLCγ antibody. Results from four fractionation experiments using FLAG-tagged constructs and two experiments using V5-tagged constructs were similar, and representative results with FLAG constructs are shown. B , representative results showing quality of fractionation by probing Western blots with anti-GAPDH, anti-Na + K + -ATPase, anti-lamin, and anti-vimentin (markers for cytosol, membrane, nuclear, and cytoskeletal fractions, respectively).

    Article Snippet: Blots were incubated with anti-TRPC3 (1:400; Alomone Laboratories, Jerusalem, Israel), anti-TRPC3-C targeted to the human C-terminal sequence RRRRLQDIEMGMGNSKSRLN (1:500 to 1:1000, Bethyl Laboratories, Inc., Montgomery, TX) , anti-TRPC3-N targeted to the murine N-terminal sequence LNGDLESAEPLERHGHKASL (1:1000; Bethyl Laboratories, Inc.) , anti-TRPC6 (1:250; Alomone Laboratories), anti-V5-HRP (1:10,000; Invitrogen), anti-FLAG (1:1000; Sigma), anti-PLCγ (SC-81, 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Epo-R (SC-697; 1:1000; Santa Cruz Biotechnology, Inc.), anti-actin (1:10,000; Sigma), anti-tubulin (1:10,000; Sigma), anti-GAPDH (1:5000; Cell Signaling Technology, Inc., Boston, MA), anti-Na+ K+ -ATPase (1:1000; Cell Signaling Technology, Inc.), anti-lamin (1:1000; Cell Signaling Technology, Inc.), anti-vimentin (1:1000; Cell Signaling Technology), or streptavidin-HRP (Pierce) antibodies.

    Techniques: Transfection, Purification, Western Blot, Construct, Fractionation

    Protein levels and activity of calpains (CAPNs) in membrane fraction of human umbilical vein endothelial cells (HUVECs) treated with arsenic trioxide (ATO). HUVECs were treated with ATO [ 0.13 μ M of arsenic (As)] for the indicated times. (A) Proteins levels of calpastatin (CAST) and CAPNs in cytosolic and membrane fractions were analyzed by western blotting. Relative protein levels in (B) cytosolic and (C) membrane fractions normalized to β - actin or Na + / K + - ATPase are shown ( n = 3 per group). (D) CAPN activity in different fractions was detected ( n = 3 per group). (E) CAPN-1 in cells treated with ATO ( 0.13 μ M As) for the indicated times was detected a using rabbit polyclonal antibody followed by goat anti-rabbit antibody (Cy3-conjuated) and nuclei were counterstained with DAPI. Representative images are shown (scale bars: 20 μ m ) and arrows indicate foci of CAPN-1 in membrane. The data are presented as the mean ± standard deviation (SD). Note: ANOVA, analysis of variance; RFU, relative fluorescence unit. * p

    Journal: Environmental Health Perspectives

    Article Title: Use of a Mouse Model and Human Umbilical Vein Endothelial Cells to Investigate the Effect of Arsenic Exposure on Vascular Endothelial Function and the Associated Role of Calpains

    doi: 10.1289/EHP4538

    Figure Lengend Snippet: Protein levels and activity of calpains (CAPNs) in membrane fraction of human umbilical vein endothelial cells (HUVECs) treated with arsenic trioxide (ATO). HUVECs were treated with ATO [ 0.13 μ M of arsenic (As)] for the indicated times. (A) Proteins levels of calpastatin (CAST) and CAPNs in cytosolic and membrane fractions were analyzed by western blotting. Relative protein levels in (B) cytosolic and (C) membrane fractions normalized to β - actin or Na + / K + - ATPase are shown ( n = 3 per group). (D) CAPN activity in different fractions was detected ( n = 3 per group). (E) CAPN-1 in cells treated with ATO ( 0.13 μ M As) for the indicated times was detected a using rabbit polyclonal antibody followed by goat anti-rabbit antibody (Cy3-conjuated) and nuclei were counterstained with DAPI. Representative images are shown (scale bars: 20 μ m ) and arrows indicate foci of CAPN-1 in membrane. The data are presented as the mean ± standard deviation (SD). Note: ANOVA, analysis of variance; RFU, relative fluorescence unit. * p

    Article Snippet: The following primary antibodies, including anti-CAST (sc-20779, Lot No. J2513, 1:500; Santa Cruz), anti-CAPN-1 (ab39170, Lot No. GR299070-9, 1:1,000; Abcam), anti-CAPN-2 (ab39165, Lot No. GR299070-9, 1:1000; Abcam), anti-CAPNS1 (ab28237, Lot No. GR640370-7, 1:1,000; Abcam), anti - β - actin (4970S, Lot No. 15, 1:1,000; Cell Signaling), and anti - Na + / K + - ATPase (3010S, Lot No. 6, 1:1,000; Cell Signaling) were diluted in 5% BSA/TBST.

    Techniques: Activity Assay, Western Blot, Standard Deviation, Fluorescence

    Cell fractionation of shortened MBP-GFP hybrid protein. Cells expressing MBP[SS128]-GFP were fractionated into inner and outer membranes as described in Materials and Methods. The immunoblot shown was decorated with anti-GFP antibody. MBP[SS128]-GFP migrated at a predicted molecular mass of 40 kDa and fractionated with the outer membrane. An apparent smaller-molecular-weight derivative of this protein was also found in the inner membrane fraction. Molecular weight (MW) standards are as shown. IM, inner membrane fraction; IM-W, KCl wash of inner membrane fraction; OM, outer membrane fraction; OM-W, KCl wash of outer membrane fraction. Although not shown, OmpA and ATPase were used to monitor fractionation of the outer membrane and inner membranes, respectively.

    Journal: Journal of Bacteriology

    Article Title: Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli †

    doi:

    Figure Lengend Snippet: Cell fractionation of shortened MBP-GFP hybrid protein. Cells expressing MBP[SS128]-GFP were fractionated into inner and outer membranes as described in Materials and Methods. The immunoblot shown was decorated with anti-GFP antibody. MBP[SS128]-GFP migrated at a predicted molecular mass of 40 kDa and fractionated with the outer membrane. An apparent smaller-molecular-weight derivative of this protein was also found in the inner membrane fraction. Molecular weight (MW) standards are as shown. IM, inner membrane fraction; IM-W, KCl wash of inner membrane fraction; OM, outer membrane fraction; OM-W, KCl wash of outer membrane fraction. Although not shown, OmpA and ATPase were used to monitor fractionation of the outer membrane and inner membranes, respectively.

    Article Snippet: Antibodies to OmpA and ATPase (Research Diagnostics, Flanders, N.J.) were used to monitor fractionation of the outer and inner membranes, respectively.

    Techniques: Cell Fractionation, Expressing, Molecular Weight, Fractionation

    SDH, mABC1, PIC, ANT, and ATPase interact with each other in co-IP studies on rat liver mitochondrial IM. IM of mitochondria was used as a control. ( A ) Co-IP of the 30-kDa component of SDH with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. ( B ) Co-IP of the 70-kDa components of SDH with Abs against 30- and 70-kDa of SDH, mABC1, ANT, ATPase, and PIC. ( C ) Co-IP of the α-subunit of ATPase with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. The α-subunit of ATPase has a molecular mass of ≈60 kDa. An Ab against the β-subunit of ATPase coimmunoprecipitated the same proteins. ( D ) Co-IP of PIC with Abs against 30- and 70-kDa component of SDH, ATPase, PIC, and mABC1. PIC has a molecular mass of ≈30 kDa. ( E ) Co-IP of mABC1 with Abs against 70-kDa components of SDH, ATPase, mABC1, and PIC. mABC1 has a molecular mass of ≈55 kDa. Negative controls included Abs against complex I and IV of the respiratory pathway, Kir6.1, and cyclophilin D. The IM of mitochondria was prepared in 1.2% digitonin and 6% lubrol WX and was dissolved in 300 mM KPi/10% ethylene glycol/5 mM EDTA/4 mM ATP/0.5 mM DTT, pH 7.9. The columns were also washed with 2–3% (octylphenoxy)polyethoxyethanol to increase the specificity of the interactions between proteins.

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

    Article Title: Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity

    doi: 10.1073/pnas.0401703101

    Figure Lengend Snippet: SDH, mABC1, PIC, ANT, and ATPase interact with each other in co-IP studies on rat liver mitochondrial IM. IM of mitochondria was used as a control. ( A ) Co-IP of the 30-kDa component of SDH with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. ( B ) Co-IP of the 70-kDa components of SDH with Abs against 30- and 70-kDa of SDH, mABC1, ANT, ATPase, and PIC. ( C ) Co-IP of the α-subunit of ATPase with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. The α-subunit of ATPase has a molecular mass of ≈60 kDa. An Ab against the β-subunit of ATPase coimmunoprecipitated the same proteins. ( D ) Co-IP of PIC with Abs against 30- and 70-kDa component of SDH, ATPase, PIC, and mABC1. PIC has a molecular mass of ≈30 kDa. ( E ) Co-IP of mABC1 with Abs against 70-kDa components of SDH, ATPase, mABC1, and PIC. mABC1 has a molecular mass of ≈55 kDa. Negative controls included Abs against complex I and IV of the respiratory pathway, Kir6.1, and cyclophilin D. The IM of mitochondria was prepared in 1.2% digitonin and 6% lubrol WX and was dissolved in 300 mM KPi/10% ethylene glycol/5 mM EDTA/4 mM ATP/0.5 mM DTT, pH 7.9. The columns were also washed with 2–3% (octylphenoxy)polyethoxyethanol to increase the specificity of the interactions between proteins.

    Article Snippet: After three rounds of booster immunization, the serum was isolated and total IgG was purified by using the protein-A purification system (Pierce). mAbs against SDH (both the 30- and 70-kDa components) and ATPase were purchased from Molecular Probes.

    Techniques: Co-Immunoprecipitation Assay

    Silver staining and Western blot analysis of the purified M-fraction. ( A ) Silver-stained SDS/PAGE gel of the mitochondrial IM extract and the purified M-fraction. We loaded ≈150 and 40 μg of protein in the IM and M-fraction lanes, respectively. ( B ) Western blots of the M-fraction with Abs against the 30- and 70-kDa components of SDH, PIC, α-subunit of ATPase, ANT, and mABC1. We loaded 40 μg of the M-fraction in each lane. The Ab against mABC1 also recognizes a smaller ≈30-kDa band.

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

    Article Title: Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity

    doi: 10.1073/pnas.0401703101

    Figure Lengend Snippet: Silver staining and Western blot analysis of the purified M-fraction. ( A ) Silver-stained SDS/PAGE gel of the mitochondrial IM extract and the purified M-fraction. We loaded ≈150 and 40 μg of protein in the IM and M-fraction lanes, respectively. ( B ) Western blots of the M-fraction with Abs against the 30- and 70-kDa components of SDH, PIC, α-subunit of ATPase, ANT, and mABC1. We loaded 40 μg of the M-fraction in each lane. The Ab against mABC1 also recognizes a smaller ≈30-kDa band.

    Article Snippet: After three rounds of booster immunization, the serum was isolated and total IgG was purified by using the protein-A purification system (Pierce). mAbs against SDH (both the 30- and 70-kDa components) and ATPase were purchased from Molecular Probes.

    Techniques: Silver Staining, Western Blot, Purification, Staining, SDS Page

    Pancreatic juice contains CD39 in the particulate fraction A , pancreatic juice collected from a pancreas stimulated with secretin and later with CCK-8 was analysed for CD39 with Western blot. Samples for analysis were collected 15–45 min after stimulation. Lanes 2 and 3 show the untreated total juice; lanes 5 and 6 show the ultracentrifuged pellet and lanes 7 and 8 show the supernatant. Agonists are indicated in each lane. In addition, lanes 1 and 4 show the Western blot of whole pancreas tissue. B , Western blot with an antibody against the α subunit of the Na + ,K + -ATPase was performed on total juice from secretin and CCK-8 stimulated pancreas (lanes 10 and 11) and as a positive control on the whole pancreas (lane 9). The gel is the representative of at least 3 different experiments.

    Journal: The Journal of Physiology

    Article Title: Rat pancreas secretes particulate ecto-nucleotidase CD39

    doi: 10.1113/jphysiol.2003.049411

    Figure Lengend Snippet: Pancreatic juice contains CD39 in the particulate fraction A , pancreatic juice collected from a pancreas stimulated with secretin and later with CCK-8 was analysed for CD39 with Western blot. Samples for analysis were collected 15–45 min after stimulation. Lanes 2 and 3 show the untreated total juice; lanes 5 and 6 show the ultracentrifuged pellet and lanes 7 and 8 show the supernatant. Agonists are indicated in each lane. In addition, lanes 1 and 4 show the Western blot of whole pancreas tissue. B , Western blot with an antibody against the α subunit of the Na + ,K + -ATPase was performed on total juice from secretin and CCK-8 stimulated pancreas (lanes 10 and 11) and as a positive control on the whole pancreas (lane 9). The gel is the representative of at least 3 different experiments.

    Article Snippet: In order to check that the 78 kDa band was not due to the plasma membrane from damaged cells that were shed into the juice during collections, we performed a Western blot with the antibody against the α subunit of the Na+ ,K+ -ATPase.

    Techniques: CCK-8 Assay, Western Blot, Positive Control

    Quantitative analyses of the expression levels of CD147 and V-ATPase in breast cancer samples. Comparison of the expression levels of (A) CD147 and (B) V-ATPase between the chemotherapy-sensitive and -resistant groups. (C) Correlation between the H-scores of CD147 and V-ATPase in breast invasive ductal cancer. ***P

    Journal: Oncology Letters

    Article Title: Cluster of differentiation 147 mediates chemoresistance in breast cancer by affecting vacuolar H+-ATPase expression and activity

    doi: 10.3892/ol.2018.8199

    Figure Lengend Snippet: Quantitative analyses of the expression levels of CD147 and V-ATPase in breast cancer samples. Comparison of the expression levels of (A) CD147 and (B) V-ATPase between the chemotherapy-sensitive and -resistant groups. (C) Correlation between the H-scores of CD147 and V-ATPase in breast invasive ductal cancer. ***P

    Article Snippet: Subsequent to washing with PBS, the cells were incubated for 2 h at room temperature with primary antibodies against CD147 (1:200; cat. no. ab212856; Abcam) and V-ATPase (1:100; cat. no. sc-69088; Santa Cruz Biotechnology, Inc. Dallas, TX, USA), followed by incubation with secondary antibodies, including anti-mouse Cy3 (1:200; cat. no. AP192C; EMD Millipore, Billerica, MA, USA) or anti-goat FITC (1:500; cat. no. ab6881; Abcam) antibodies for 1 h at room temperature.

    Techniques: Expressing

    Immunohistochemical observation of CD147 and V-ATPase expression in chemotherapy-sensitive or -resistant breast invasive ductal cancer patients (magnification, ×400). Chemotherapy-sensitive patients exhibited moderate expression of (A) CD147 and (B) V-ATPase in the cell membrane. Chemotherapy-resistant patients exhibited high expression of (C) CD147 in the cell membrane and (D) V-ATPase mainly in the cell membrane and cytoplasm. CD147, cluster of differentiation 147; V-ATPase, vacuolar H + -ATPase.

    Journal: Oncology Letters

    Article Title: Cluster of differentiation 147 mediates chemoresistance in breast cancer by affecting vacuolar H+-ATPase expression and activity

    doi: 10.3892/ol.2018.8199

    Figure Lengend Snippet: Immunohistochemical observation of CD147 and V-ATPase expression in chemotherapy-sensitive or -resistant breast invasive ductal cancer patients (magnification, ×400). Chemotherapy-sensitive patients exhibited moderate expression of (A) CD147 and (B) V-ATPase in the cell membrane. Chemotherapy-resistant patients exhibited high expression of (C) CD147 in the cell membrane and (D) V-ATPase mainly in the cell membrane and cytoplasm. CD147, cluster of differentiation 147; V-ATPase, vacuolar H + -ATPase.

    Article Snippet: Subsequent to washing with PBS, the cells were incubated for 2 h at room temperature with primary antibodies against CD147 (1:200; cat. no. ab212856; Abcam) and V-ATPase (1:100; cat. no. sc-69088; Santa Cruz Biotechnology, Inc. Dallas, TX, USA), followed by incubation with secondary antibodies, including anti-mouse Cy3 (1:200; cat. no. AP192C; EMD Millipore, Billerica, MA, USA) or anti-goat FITC (1:500; cat. no. ab6881; Abcam) antibodies for 1 h at room temperature.

    Techniques: Immunohistochemistry, Expressing

    Immunofluorescence was used to detect CD147 and V-ATPase expression and subcellular localization in the four transfected cell lines. Experiments were repeated at least three times, and a representative experiment is shown. CD147, cluster of differentiation 147; V-ATPase, vacuolar H + -ATPase; CON, control; CD, CD147 overexpression; si, CD147 knockdown. Blue represents nuclear staining; red represents CD147 staining; yellow represents V-ATPase staining.

    Journal: Oncology Letters

    Article Title: Cluster of differentiation 147 mediates chemoresistance in breast cancer by affecting vacuolar H+-ATPase expression and activity

    doi: 10.3892/ol.2018.8199

    Figure Lengend Snippet: Immunofluorescence was used to detect CD147 and V-ATPase expression and subcellular localization in the four transfected cell lines. Experiments were repeated at least three times, and a representative experiment is shown. CD147, cluster of differentiation 147; V-ATPase, vacuolar H + -ATPase; CON, control; CD, CD147 overexpression; si, CD147 knockdown. Blue represents nuclear staining; red represents CD147 staining; yellow represents V-ATPase staining.

    Article Snippet: Subsequent to washing with PBS, the cells were incubated for 2 h at room temperature with primary antibodies against CD147 (1:200; cat. no. ab212856; Abcam) and V-ATPase (1:100; cat. no. sc-69088; Santa Cruz Biotechnology, Inc. Dallas, TX, USA), followed by incubation with secondary antibodies, including anti-mouse Cy3 (1:200; cat. no. AP192C; EMD Millipore, Billerica, MA, USA) or anti-goat FITC (1:500; cat. no. ab6881; Abcam) antibodies for 1 h at room temperature.

    Techniques: Immunofluorescence, Expressing, Transfection, Over Expression, Staining

    Western blot of whole cell (WC) homogenates and apical membrane fractions from two immortalized hRPTC lines grown in Transwell membranes. Arrows on the left-hand side of the blots indicate the molecular sizes, while the arrows on the right-hand side indicate the molecular sizes of the bands of the proteins of interest. The blot was probed for NBCe2 along with the following membrane markers: CD-13 (APN microvilli marker), Na + ,K + /ATPase (basolateral membrane marker), and NBCe1 (basolateral membrane marker). The results demonstrate that two isoforms of NBCe2 exist in the apical membrane of polarized RPTCs grown on Transwells. The control western blots demonstrate that αNa + ,K + /ATPase (NKA) and NBCe1 only stain weakly in the apical fraction, probably due to some basolateral contamination inherent in this kind of preparation. WC homogenates demonstrate the presence of NBCe2, NBCe1, CD-13, and Na + ,K + /ATPase, as expected.

    Journal: PLoS ONE

    Article Title: Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells

    doi: 10.1371/journal.pone.0189464

    Figure Lengend Snippet: Western blot of whole cell (WC) homogenates and apical membrane fractions from two immortalized hRPTC lines grown in Transwell membranes. Arrows on the left-hand side of the blots indicate the molecular sizes, while the arrows on the right-hand side indicate the molecular sizes of the bands of the proteins of interest. The blot was probed for NBCe2 along with the following membrane markers: CD-13 (APN microvilli marker), Na + ,K + /ATPase (basolateral membrane marker), and NBCe1 (basolateral membrane marker). The results demonstrate that two isoforms of NBCe2 exist in the apical membrane of polarized RPTCs grown on Transwells. The control western blots demonstrate that αNa + ,K + /ATPase (NKA) and NBCe1 only stain weakly in the apical fraction, probably due to some basolateral contamination inherent in this kind of preparation. WC homogenates demonstrate the presence of NBCe2, NBCe1, CD-13, and Na + ,K + /ATPase, as expected.

    Article Snippet: The blot was probed with NBCe2 [ ], CD-13 (APN), Na+ ,K+ /ATPase, PAT-1, and NBCe1 antibodies.

    Techniques: Western Blot, Marker, Staining

    Models of ion transport in hRPTCs. This is a model of the hRPTC with the apical (brush border, facing the lumen) (left hand side) and the basolateral side (right hand side). The principal ion transporters and some of the receptors that regulate them are shown. Starting at 11 o’clock in blue is shown the classic pathway for transporting bicarbonate (HCO 3 - ) into the cell. Filtered NaHCO 3 dissociates into Na + and HCO 3 . HCO 3 - in the luminal fluid and H + secreted into the lumen form H 2 CO 3 . Carbonic anhydrase type 4 (CA IV) in the luminal membrane catalyzes the conversion of H 2 CO 3 to H 2 O and CO 2 .CO 2 diffuses inside the hRPTC where intracellular carbonic anhydrase type 2 (CA II) catalyzes the conversion of CO 2 and H 2 O into H 2 CO 3 which then dissociates into HCO 3 - and H + . At 9 o’clock is NHE3 which exchanges one Na + from the lumen with one H + inside the hRPTC. At 7 o’clock HCO 3 - Cl - exchanger (PAT1) is depicted which exchanges luminal Cl - with cytoplasmic HCO 3 - . At 3 o’clock is depicted NBCe1 at the basolateral membrane which electrogenically transports 2–3 Na + and one HCO 3 - into the basolateral space. At 4 o’clock is Na + , K + /ATPase which pumps 3 Na + out of the cell into the blood stream and pumps in 2 K + inside the cell The topic of this manuscript deals with NBCe2, drawn at 8 o’clock. Under a normal sodium load it plays a minor role in Na + and HCO 3 - transport into the hRPTC. There are various plasma membrane receptors that regulate some of these transporters/ exchanger/pumps. The dopamine-1 receptor (D 1 R) (ten o’clock) when stimulated with dopamine (green box) inhibits (red lines) both NHE3 and Na + , K + /ATPase (without the red line, for simplicity) activities resulting in reduced Na + reabsorption and increased Na + excretion. The AT 1 R (5 o’clock) increases Na + , K + /ATPase activity (green arrow) resulting in increased Na + reabsorption. An increase in intracellular Na + increases Na + , K + /ATPase activity (5 o’clock) that is abetted by AT 1 R (green arrow) resulting in increased Na + transport from inside the cell to the basolateral space. The D 1 R and AT 1 R oppose each other. The D 1 R inhibits the AT 1 R, resulting in reduced Na + transport. We showed that NBCe2 is not affected by stimulation of the D 1 R or AT 1 R. Under basal conditions, NBCe1 is more active than NBCe2 (depicted as relatively larger directional transport arrows).

    Journal: PLoS ONE

    Article Title: Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells

    doi: 10.1371/journal.pone.0189464

    Figure Lengend Snippet: Models of ion transport in hRPTCs. This is a model of the hRPTC with the apical (brush border, facing the lumen) (left hand side) and the basolateral side (right hand side). The principal ion transporters and some of the receptors that regulate them are shown. Starting at 11 o’clock in blue is shown the classic pathway for transporting bicarbonate (HCO 3 - ) into the cell. Filtered NaHCO 3 dissociates into Na + and HCO 3 . HCO 3 - in the luminal fluid and H + secreted into the lumen form H 2 CO 3 . Carbonic anhydrase type 4 (CA IV) in the luminal membrane catalyzes the conversion of H 2 CO 3 to H 2 O and CO 2 .CO 2 diffuses inside the hRPTC where intracellular carbonic anhydrase type 2 (CA II) catalyzes the conversion of CO 2 and H 2 O into H 2 CO 3 which then dissociates into HCO 3 - and H + . At 9 o’clock is NHE3 which exchanges one Na + from the lumen with one H + inside the hRPTC. At 7 o’clock HCO 3 - Cl - exchanger (PAT1) is depicted which exchanges luminal Cl - with cytoplasmic HCO 3 - . At 3 o’clock is depicted NBCe1 at the basolateral membrane which electrogenically transports 2–3 Na + and one HCO 3 - into the basolateral space. At 4 o’clock is Na + , K + /ATPase which pumps 3 Na + out of the cell into the blood stream and pumps in 2 K + inside the cell The topic of this manuscript deals with NBCe2, drawn at 8 o’clock. Under a normal sodium load it plays a minor role in Na + and HCO 3 - transport into the hRPTC. There are various plasma membrane receptors that regulate some of these transporters/ exchanger/pumps. The dopamine-1 receptor (D 1 R) (ten o’clock) when stimulated with dopamine (green box) inhibits (red lines) both NHE3 and Na + , K + /ATPase (without the red line, for simplicity) activities resulting in reduced Na + reabsorption and increased Na + excretion. The AT 1 R (5 o’clock) increases Na + , K + /ATPase activity (green arrow) resulting in increased Na + reabsorption. An increase in intracellular Na + increases Na + , K + /ATPase activity (5 o’clock) that is abetted by AT 1 R (green arrow) resulting in increased Na + transport from inside the cell to the basolateral space. The D 1 R and AT 1 R oppose each other. The D 1 R inhibits the AT 1 R, resulting in reduced Na + transport. We showed that NBCe2 is not affected by stimulation of the D 1 R or AT 1 R. Under basal conditions, NBCe1 is more active than NBCe2 (depicted as relatively larger directional transport arrows).

    Article Snippet: The blot was probed with NBCe2 [ ], CD-13 (APN), Na+ ,K+ /ATPase, PAT-1, and NBCe1 antibodies.

    Techniques: Activity Assay

    The effect of BI6015 on the expression of Na + ,K + /ATPase (NKA) and the putative anion transporter type 1 (PAT-1). Compared to vehicle control (VEH), neither NKA nor PAT-1 is affected by the addition of the HNF4A inhibitor BI6015. α tubulin was used as a protein loading control.

    Journal: PLoS ONE

    Article Title: Sodium bicarbonate cotransporter NBCe2 gene variants increase sodium and bicarbonate transport in human renal proximal tubule cells

    doi: 10.1371/journal.pone.0189464

    Figure Lengend Snippet: The effect of BI6015 on the expression of Na + ,K + /ATPase (NKA) and the putative anion transporter type 1 (PAT-1). Compared to vehicle control (VEH), neither NKA nor PAT-1 is affected by the addition of the HNF4A inhibitor BI6015. α tubulin was used as a protein loading control.

    Article Snippet: The blot was probed with NBCe2 [ ], CD-13 (APN), Na+ ,K+ /ATPase, PAT-1, and NBCe1 antibodies.

    Techniques: Expressing

    Effect of vanadate on renal α 1 -Na, K-ATPase protein abundance. Ten days of vanadate injection significantly suppressed α 1 -Na, K-ATPase protein abundance. Histogram bars show the densitometric analyses ratios of α 1 -Na, K-ATPase to β-actin intensity, and the representative immunoblot photographs are present. All values are expressed as the mean ± SD of 8 rats/group. *Significantly different from the sham group ( p

    Journal: Toxicological Research

    Article Title: Vanadate-Induced Renal cAMP and Malondialdehyde Accumulation Suppresses Alpha 1 Sodium Potassium Adenosine Triphosphatase Protein Levels

    doi: 10.5487/TR.2018.34.2.143

    Figure Lengend Snippet: Effect of vanadate on renal α 1 -Na, K-ATPase protein abundance. Ten days of vanadate injection significantly suppressed α 1 -Na, K-ATPase protein abundance. Histogram bars show the densitometric analyses ratios of α 1 -Na, K-ATPase to β-actin intensity, and the representative immunoblot photographs are present. All values are expressed as the mean ± SD of 8 rats/group. *Significantly different from the sham group ( p

    Article Snippet: Cyclic AMP phosphorylates Ser943 on the α subunit of Na, K-ATPase and reduces its activity in the rat TALH ( , ).

    Techniques: Injection

    Representative immunohistochemical staining images of renal α 1 -Na, K-ATPase protein localization in the cortex (A, D), outer medulla (B, E), and inner medulla (C, F) from sham (A–C), and vanadate (D–F) (n = 5/group). Vanadate reduced immunoreactivity both in the cortex (PCT and Pcap; arrows) and the medulla (PTs and MCD; arrow heads). Bar scale = 20 μm.

    Journal: Toxicological Research

    Article Title: Vanadate-Induced Renal cAMP and Malondialdehyde Accumulation Suppresses Alpha 1 Sodium Potassium Adenosine Triphosphatase Protein Levels

    doi: 10.5487/TR.2018.34.2.143

    Figure Lengend Snippet: Representative immunohistochemical staining images of renal α 1 -Na, K-ATPase protein localization in the cortex (A, D), outer medulla (B, E), and inner medulla (C, F) from sham (A–C), and vanadate (D–F) (n = 5/group). Vanadate reduced immunoreactivity both in the cortex (PCT and Pcap; arrows) and the medulla (PTs and MCD; arrow heads). Bar scale = 20 μm.

    Article Snippet: Cyclic AMP phosphorylates Ser943 on the α subunit of Na, K-ATPase and reduces its activity in the rat TALH ( , ).

    Techniques: Immunohistochemistry, Staining

    Intracellular localization of subunit a3 and markers for the Golgi apparatus and lysosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to the Golgi and lysosomes, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and Giantin or LAMP2 as markers for the Golgi and lysosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the Golgi apparatus marker Giantin ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the lysosomal marker LAMP2 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.

    Journal: Oncotarget

    Article Title: The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

    doi: 10.18632/oncotarget.10063

    Figure Lengend Snippet: Intracellular localization of subunit a3 and markers for the Golgi apparatus and lysosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to the Golgi and lysosomes, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and Giantin or LAMP2 as markers for the Golgi and lysosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the Golgi apparatus marker Giantin ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the lysosomal marker LAMP2 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.

    Article Snippet: Briefly, an siRNA pool specific for the subunit a3 isoform of the V-ATPase was purchased from Dharmacon.

    Techniques: Incubation, Staining, Marker, Labeling

    Subunit a3 colocalizes with a leading edge marker in migrating breast cancer cells MCF10CA1a, MB231, and SUM149 breast cancer cells were grown to confluence on poly-d-lysine coated coverslips. A wound was made in the cell monolayer and cells were allowed to migrate for 4 h in order to induce the formation of a leading edge. Cells were then fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and the leading edge marker cortactin followed by incubation with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. Shown are representative images of MCF10CA1a, MB231, and SUM149 cells stained for cortactin ( left ), a3 ( second from left ), and DAPI ( second from right ). The merged images are present to the far right. White arrows indicate subunit a3 and cortactin colocalization at the leading edge. The images depict representative staining from a minimum of two separate experiments, with 50-80 cells imaged per experiment for each condition tested.

    Journal: Oncotarget

    Article Title: The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

    doi: 10.18632/oncotarget.10063

    Figure Lengend Snippet: Subunit a3 colocalizes with a leading edge marker in migrating breast cancer cells MCF10CA1a, MB231, and SUM149 breast cancer cells were grown to confluence on poly-d-lysine coated coverslips. A wound was made in the cell monolayer and cells were allowed to migrate for 4 h in order to induce the formation of a leading edge. Cells were then fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and the leading edge marker cortactin followed by incubation with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. Shown are representative images of MCF10CA1a, MB231, and SUM149 cells stained for cortactin ( left ), a3 ( second from left ), and DAPI ( second from right ). The merged images are present to the far right. White arrows indicate subunit a3 and cortactin colocalization at the leading edge. The images depict representative staining from a minimum of two separate experiments, with 50-80 cells imaged per experiment for each condition tested.

    Article Snippet: Briefly, an siRNA pool specific for the subunit a3 isoform of the V-ATPase was purchased from Dharmacon.

    Techniques: Marker, Incubation, Staining

    Intracellular localization of subunit a3 and markers for early and late endosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to endosomal compartments, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and EEA1 or Rab7 as markers for early endosomes and late endosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the early endosome marker EEA1 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the late endosome marker Rab7 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.

    Journal: Oncotarget

    Article Title: The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

    doi: 10.18632/oncotarget.10063

    Figure Lengend Snippet: Intracellular localization of subunit a3 and markers for early and late endosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to endosomal compartments, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and EEA1 or Rab7 as markers for early endosomes and late endosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the early endosome marker EEA1 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the late endosome marker Rab7 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.

    Article Snippet: Briefly, an siRNA pool specific for the subunit a3 isoform of the V-ATPase was purchased from Dharmacon.

    Techniques: Incubation, Staining, Marker, Labeling

    Subunit a3 localizes to the plasma membrane of human breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown to confluence on poly-d-lysine coated coverslips. To assess the effects of cell migration on a3 localization, a wound was made in the cell monolayer and cells were allowed to migrate for 4 h. Cells were then fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and Alexa Fluor® 568 phalloidin to stain actin followed by incubation with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Shown are representative images of confluent MCF10a, MCF10CA1a, MB231, and SUM149 cells stained for phalloidin ( left ), subunit a3 (second from left ), DAPI ( second from right ), and the merged images ( right ). B. Shown are representative images of MCF10a, MCF10CA1a, MB231, and SUM149 cells (with monolayer wounded) stained for phalloidin ( left ), subunit a3 (second from left ), DAPI ( second from right ), and the merged images ( right ). White arrows indicate plasma membrane subunit a3. C. Quantification of plasma membrane staining for subunit a3 in confluent or migrating cells. To quantitate plasma membrane staining, 50-80 cells from three separate experiments were counted and the number of cells showing plasma membrane a3 localization was determined. The graphed data represents the percentage of cells showing a3 localization to the plasma membrane. All error bars indicate S.E. *, p

    Journal: Oncotarget

    Article Title: The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

    doi: 10.18632/oncotarget.10063

    Figure Lengend Snippet: Subunit a3 localizes to the plasma membrane of human breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown to confluence on poly-d-lysine coated coverslips. To assess the effects of cell migration on a3 localization, a wound was made in the cell monolayer and cells were allowed to migrate for 4 h. Cells were then fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and Alexa Fluor® 568 phalloidin to stain actin followed by incubation with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Shown are representative images of confluent MCF10a, MCF10CA1a, MB231, and SUM149 cells stained for phalloidin ( left ), subunit a3 (second from left ), DAPI ( second from right ), and the merged images ( right ). B. Shown are representative images of MCF10a, MCF10CA1a, MB231, and SUM149 cells (with monolayer wounded) stained for phalloidin ( left ), subunit a3 (second from left ), DAPI ( second from right ), and the merged images ( right ). White arrows indicate plasma membrane subunit a3. C. Quantification of plasma membrane staining for subunit a3 in confluent or migrating cells. To quantitate plasma membrane staining, 50-80 cells from three separate experiments were counted and the number of cells showing plasma membrane a3 localization was determined. The graphed data represents the percentage of cells showing a3 localization to the plasma membrane. All error bars indicate S.E. *, p

    Article Snippet: Briefly, an siRNA pool specific for the subunit a3 isoform of the V-ATPase was purchased from Dharmacon.

    Techniques: Migration, Staining, Incubation

    STED microscopy dissecting the localization of Na + ,K + -ATPase in cultured striatal neurons . The 5 × 3 mosaic shows a comparision of the confocal (left) and STED images (middle) of the Alexa-594 immunolabelled α3 NKA in dendritic spines. Postprocessing of the raw STED data by a Richardson-Lucy deconvolution further enhances the details as shown in the right row of images. Scale bar: 500 nm

    Journal: BMC Neuroscience

    Article Title: Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy

    doi: 10.1186/1471-2202-12-16

    Figure Lengend Snippet: STED microscopy dissecting the localization of Na + ,K + -ATPase in cultured striatal neurons . The 5 × 3 mosaic shows a comparision of the confocal (left) and STED images (middle) of the Alexa-594 immunolabelled α3 NKA in dendritic spines. Postprocessing of the raw STED data by a Richardson-Lucy deconvolution further enhances the details as shown in the right row of images. Scale bar: 500 nm

    Article Snippet: Antibodies In both imaging and biochemical assays the well characterized mouse monoclonal anti-α3 Na + ,K+ -ATPase (Affinity bioreagents, MA3-915) was used [ ].

    Techniques: Microscopy, Cell Culture

    Immunostaining of MCF10a cells overexpressing subunit a isoforms using an antibody against V-ATPase. MCF10a cells were grown as a monolayer on coverslips in 6-well plates. Cells were immunostained using an antibody against subunit A of V-ATPase. Images

    Journal: The Journal of Biological Chemistry

    Article Title: The Function of Vacuolar ATPase (V-ATPase) a Subunit Isoforms in Invasiveness of MCF10a and MCF10CA1a Human Breast Cancer Cells *

    doi: 10.1074/jbc.M113.503771

    Figure Lengend Snippet: Immunostaining of MCF10a cells overexpressing subunit a isoforms using an antibody against V-ATPase. MCF10a cells were grown as a monolayer on coverslips in 6-well plates. Cells were immunostained using an antibody against subunit A of V-ATPase. Images

    Article Snippet: A monoclonal antibody raised against the V-ATPase subunit A (Sigma, clone 4F5) was used to determine the in situ localization of V-ATPases.

    Techniques: Immunostaining

    Immunostaining of MCF10a and MCF10CA1a cells using an antibody against V-ATPase. MCF10a and MCF10CA1a cells were grown as a monolayer on coverslips in 6-well plates. Cells were immunostained using an antibody against subunit A of V-ATPase (part of the

    Journal: The Journal of Biological Chemistry

    Article Title: The Function of Vacuolar ATPase (V-ATPase) a Subunit Isoforms in Invasiveness of MCF10a and MCF10CA1a Human Breast Cancer Cells *

    doi: 10.1074/jbc.M113.503771

    Figure Lengend Snippet: Immunostaining of MCF10a and MCF10CA1a cells using an antibody against V-ATPase. MCF10a and MCF10CA1a cells were grown as a monolayer on coverslips in 6-well plates. Cells were immunostained using an antibody against subunit A of V-ATPase (part of the

    Article Snippet: A monoclonal antibody raised against the V-ATPase subunit A (Sigma, clone 4F5) was used to determine the in situ localization of V-ATPases.

    Techniques: Immunostaining

    Profiling of membrane-fraction proteins from the prefrontal cortex of control and alcoholic subjects. Control (C) or alcoholic (A) prefrontal cortex membrane fraction proteins were resolved by 1D PAGE and proteins stained with colloidal Coomassie. Alcoholic subjects displayed a prominent reduction in the level of protein staining at a protein band of ∼270 kDa and upregulation at ∼112 kDa (upper panel). Protein levels of transmembranous (∼112 kDa) Na + ,K + -ATPase α-subunit, and oligomeric forms of the α-subunit were visualized by Western blotting (middle panel). The level of transmembranous α-subunit was quantified using actin for normalisation (lower panel). For marked significance: *** = p

    Journal: PLoS ONE

    Article Title: Alcohol-Related Brain Damage in Humans

    doi: 10.1371/journal.pone.0093586

    Figure Lengend Snippet: Profiling of membrane-fraction proteins from the prefrontal cortex of control and alcoholic subjects. Control (C) or alcoholic (A) prefrontal cortex membrane fraction proteins were resolved by 1D PAGE and proteins stained with colloidal Coomassie. Alcoholic subjects displayed a prominent reduction in the level of protein staining at a protein band of ∼270 kDa and upregulation at ∼112 kDa (upper panel). Protein levels of transmembranous (∼112 kDa) Na + ,K + -ATPase α-subunit, and oligomeric forms of the α-subunit were visualized by Western blotting (middle panel). The level of transmembranous α-subunit was quantified using actin for normalisation (lower panel). For marked significance: *** = p

    Article Snippet: Both protein doublet bands at ∼112 kDa protein were identified as the α3 subunit of the Na+ ,K+ -ATPase – refer to , and .

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Western Blot

    Electron microscopical analysis of caveolin-1 in cultured human ARPE-19 cells. (A) Micrograph of an ARPE-19 cell depicting apical microvilli (AM) and basal infoldings (BI). The nucleus (N) can also be seen (7700×). Panel A serves as a comparison for panels B–E, which are higher magnifications of the apical and basal plasma membranes of a RPE cell. (B) Higher magnification of apical microvilli labelled with an antibody against caveolin-1. No gold particles were observed (60,000×). (C) Higher magnification of the basal infoldings of RPE labelled with an antibody against caveolin-1. Arrows point to gold particles (35,500×). (D) Higher magnification of apical microvilli labelled with antibody against the Na + ,K + -ATPase. Gold particles were abundant (46,000×). (E) Higher magnification of the basolateral infoldings of A RPE cells labelled with an antibody against folate receptor α . Gold particles were present across the membrane (46,000×).

    Journal: The Histochemical journal

    Article Title: A comparison of caveolae and caveolin-1 to folate receptor α in retina and retinal pigment epithelium

    doi:

    Figure Lengend Snippet: Electron microscopical analysis of caveolin-1 in cultured human ARPE-19 cells. (A) Micrograph of an ARPE-19 cell depicting apical microvilli (AM) and basal infoldings (BI). The nucleus (N) can also be seen (7700×). Panel A serves as a comparison for panels B–E, which are higher magnifications of the apical and basal plasma membranes of a RPE cell. (B) Higher magnification of apical microvilli labelled with an antibody against caveolin-1. No gold particles were observed (60,000×). (C) Higher magnification of the basal infoldings of RPE labelled with an antibody against caveolin-1. Arrows point to gold particles (35,500×). (D) Higher magnification of apical microvilli labelled with antibody against the Na + ,K + -ATPase. Gold particles were abundant (46,000×). (E) Higher magnification of the basolateral infoldings of A RPE cells labelled with an antibody against folate receptor α . Gold particles were present across the membrane (46,000×).

    Article Snippet: After blocking with 4% normal goat serum, the sections were incubated for 6 h at room temperature with a polyclonal IgG against caveolin-1 (1 : 100), a polyclonal IgG against folate receptor α (1 : 50), or a polyclonal antibody against Na+ ,K+ -ATPase (1 : 50).

    Techniques: Cell Culture

    Laser scanning confocal microscopic localization of caveolin-1 and folate receptor α in intact mouse RPE. (A) High magnification of mouse retina incubated with an antibody against caveolin-1. The apical and basal plasma membranes of the RPE are represented by the top (a) and bottom (b) two-headed arrows, respectively. Caveolin-1 is present on the apical (arrows) and basal (arrowheads) plasmalemmal surfaces of the RPE cell. (B) High magnification of mouse retina incubated with an antibody against the folate receptor α . Note the intense bands of fluorescence on the basolateral membrane of RPE, but no immunoreactivity in the apical region. (C) High magnification of mouse retina incubated with an antibody against the Na + ,K + -ATPase. As expected, an intense band of fluorescent labelling was observed on the apical surface of the RPE monolayer. (D) Haematoxylin and eosin-stained section of mouse retina shown for comparison (630×). Abbreviations: IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium.

    Journal: The Histochemical journal

    Article Title: A comparison of caveolae and caveolin-1 to folate receptor α in retina and retinal pigment epithelium

    doi:

    Figure Lengend Snippet: Laser scanning confocal microscopic localization of caveolin-1 and folate receptor α in intact mouse RPE. (A) High magnification of mouse retina incubated with an antibody against caveolin-1. The apical and basal plasma membranes of the RPE are represented by the top (a) and bottom (b) two-headed arrows, respectively. Caveolin-1 is present on the apical (arrows) and basal (arrowheads) plasmalemmal surfaces of the RPE cell. (B) High magnification of mouse retina incubated with an antibody against the folate receptor α . Note the intense bands of fluorescence on the basolateral membrane of RPE, but no immunoreactivity in the apical region. (C) High magnification of mouse retina incubated with an antibody against the Na + ,K + -ATPase. As expected, an intense band of fluorescent labelling was observed on the apical surface of the RPE monolayer. (D) Haematoxylin and eosin-stained section of mouse retina shown for comparison (630×). Abbreviations: IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium.

    Article Snippet: After blocking with 4% normal goat serum, the sections were incubated for 6 h at room temperature with a polyclonal IgG against caveolin-1 (1 : 100), a polyclonal IgG against folate receptor α (1 : 50), or a polyclonal antibody against Na+ ,K+ -ATPase (1 : 50).

    Techniques: Incubation, Fluorescence, Staining

    Electron microscopical immunohistochemical analysis of caveolin-1 in mammalian retina. (A) Mouse RPE depicting apical microvilli (AM) and basal infoldings (BI). The nucleus (N) and mitochondria (M) are also visible (7700×). Panel A serves as a comparison for panels B–E, which are higher magnifications of the apical and basal plasma membranes of a RPE cell. (B) Higher magnification of apical microvilli labelled with an antibody against caveolin-1. Arrows point to gold particles indicative of positive labelling (27,500×). (C) Higher magnification of the basal infoldings of RPE labelled with an antibody against caveolin-1. Arrows point to gold particles (35,500×). (D) Higher magnification of apical microvilli labelled with antibody against the Na + ,K + -ATPase (60,000×). Gold particles were abundant. (E) Higher magnification of the basolateral infoldings of a RPE cell labelled with an antibody against folate receptor α (60,000×). Gold particles were present across the membrane.

    Journal: The Histochemical journal

    Article Title: A comparison of caveolae and caveolin-1 to folate receptor α in retina and retinal pigment epithelium

    doi:

    Figure Lengend Snippet: Electron microscopical immunohistochemical analysis of caveolin-1 in mammalian retina. (A) Mouse RPE depicting apical microvilli (AM) and basal infoldings (BI). The nucleus (N) and mitochondria (M) are also visible (7700×). Panel A serves as a comparison for panels B–E, which are higher magnifications of the apical and basal plasma membranes of a RPE cell. (B) Higher magnification of apical microvilli labelled with an antibody against caveolin-1. Arrows point to gold particles indicative of positive labelling (27,500×). (C) Higher magnification of the basal infoldings of RPE labelled with an antibody against caveolin-1. Arrows point to gold particles (35,500×). (D) Higher magnification of apical microvilli labelled with antibody against the Na + ,K + -ATPase (60,000×). Gold particles were abundant. (E) Higher magnification of the basolateral infoldings of a RPE cell labelled with an antibody against folate receptor α (60,000×). Gold particles were present across the membrane.

    Article Snippet: After blocking with 4% normal goat serum, the sections were incubated for 6 h at room temperature with a polyclonal IgG against caveolin-1 (1 : 100), a polyclonal IgG against folate receptor α (1 : 50), or a polyclonal antibody against Na+ ,K+ -ATPase (1 : 50).

    Techniques: Immunohistochemistry

    Identification of galactose as a hypoglycemic compound using the hyperglycemic silkworm model. (A) Preparation protocol for the jiou extract. (B) Silkworms were fed a 10% (w/w) glucose diet for 60 min. 50 µl of Jiou extract (1 mg/ml) or human insulin (3.5 mg/ml) was injected into the hemolymph of the hyperglycemic silkworms. The silkworms were fasted for 6 h and the sugar level in the hemolymph was determined. n = 6–7 per group. Data are shown as means ± SD. (C, D) The jiou extract was treated with TFA, and analyzed by TLC. Sugars were localized with 10% sulfuric acid solution. (E) Silkworms were fed a 10% (w/w) glucose diet for 60 min. 50 µl of D-Galactose (Gal), D-glucose (Glu), D-talose (Tal), D-mannose (Man), (1 mg/ml) or human insulin (3.5 mg/ml) was injected into the hemolymph of hyperglycemic silkworms. Silkworms were fasted for 6 h and sugar level in the hemolymph was determined. n = 6 per group. (F) Structure of sugar is shown by Fischer projection in the panel. Numbers shown on the left indicate the carbon positions of the sugar. Red hydroxyl group indicates the positions that differ from D-galactose. Activity represents the hypoglycemic effect. Data are shown as means ± SD. NS; not significant. (G) Galactose (10 mg/ml, 0.5 ml i.p.) was injected to streptozotocin induced-diabetic mice, and blood glucose level was determined after 4 h of fasting. Blood glucose levels in streptozotocin-induced hyperglycemic mice were measured (blood glucose 250–400 mg/dl) and then the mice were treated with either PBS or galactose solution. Four hours after administration and the removal of diet, the blood glucose levels were measured again. The data represent the blood glucose value after treatment divided by the blood glucose value before treatment of individual animals. In all panels, the statistical significance of the difference was evaluated using Student's t test. (H) Blood glucose levels in streptozotocin-induced hyperglycemic mice were measured (blood glucose 250–400 mg/dl) and then the mice were treated with either PBS, galactose (200 mg/kg mouse, i.p.), or glucose (200 mg/kg mouse, i.p.) solution. Two hours after administration and removal of the diet, the mice were killed and the membrane fraction in mouse liver was isolated. GLUT2 and Na, K-ATPase were detected by Western blot analysis with anti-GLUT2 antibody or anti-Na, K-ATPase antibody. Immunoblots of GLUT2 and Na, K-ATPase (Top) and calculations of relative GLUT2 (Bottom). n = 3–4 per group. Data at the bottom of the figure are shown as means ± SD. In all panels, the statistical significance of the difference was evaluated using Student's t test.

    Journal: PLoS ONE

    Article Title: An Invertebrate Hyperglycemic Model for the Identification of Anti-Diabetic Drugs

    doi: 10.1371/journal.pone.0018292

    Figure Lengend Snippet: Identification of galactose as a hypoglycemic compound using the hyperglycemic silkworm model. (A) Preparation protocol for the jiou extract. (B) Silkworms were fed a 10% (w/w) glucose diet for 60 min. 50 µl of Jiou extract (1 mg/ml) or human insulin (3.5 mg/ml) was injected into the hemolymph of the hyperglycemic silkworms. The silkworms were fasted for 6 h and the sugar level in the hemolymph was determined. n = 6–7 per group. Data are shown as means ± SD. (C, D) The jiou extract was treated with TFA, and analyzed by TLC. Sugars were localized with 10% sulfuric acid solution. (E) Silkworms were fed a 10% (w/w) glucose diet for 60 min. 50 µl of D-Galactose (Gal), D-glucose (Glu), D-talose (Tal), D-mannose (Man), (1 mg/ml) or human insulin (3.5 mg/ml) was injected into the hemolymph of hyperglycemic silkworms. Silkworms were fasted for 6 h and sugar level in the hemolymph was determined. n = 6 per group. (F) Structure of sugar is shown by Fischer projection in the panel. Numbers shown on the left indicate the carbon positions of the sugar. Red hydroxyl group indicates the positions that differ from D-galactose. Activity represents the hypoglycemic effect. Data are shown as means ± SD. NS; not significant. (G) Galactose (10 mg/ml, 0.5 ml i.p.) was injected to streptozotocin induced-diabetic mice, and blood glucose level was determined after 4 h of fasting. Blood glucose levels in streptozotocin-induced hyperglycemic mice were measured (blood glucose 250–400 mg/dl) and then the mice were treated with either PBS or galactose solution. Four hours after administration and the removal of diet, the blood glucose levels were measured again. The data represent the blood glucose value after treatment divided by the blood glucose value before treatment of individual animals. In all panels, the statistical significance of the difference was evaluated using Student's t test. (H) Blood glucose levels in streptozotocin-induced hyperglycemic mice were measured (blood glucose 250–400 mg/dl) and then the mice were treated with either PBS, galactose (200 mg/kg mouse, i.p.), or glucose (200 mg/kg mouse, i.p.) solution. Two hours after administration and removal of the diet, the mice were killed and the membrane fraction in mouse liver was isolated. GLUT2 and Na, K-ATPase were detected by Western blot analysis with anti-GLUT2 antibody or anti-Na, K-ATPase antibody. Immunoblots of GLUT2 and Na, K-ATPase (Top) and calculations of relative GLUT2 (Bottom). n = 3–4 per group. Data at the bottom of the figure are shown as means ± SD. In all panels, the statistical significance of the difference was evaluated using Student's t test.

    Article Snippet: The relative amount of phosphorylated Akt or phosphorylated AMPK or GLUT2 on total Akt or total AMPK or Na, K-ATPase was determined.

    Techniques: Injection, Thin Layer Chromatography, Activity Assay, Mouse Assay, Isolation, Western Blot

    The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + -ATPase (NKA), and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P

    Journal: Frontiers in Physiology

    Article Title: The Antioxidant Peroxiredoxin 6 (Prdx6) Exhibits Different Profiles in the Livers of Seawater- and Fresh Water-Acclimated Milkfish, Chanos chanos, upon Hypothermal Challenge

    doi: 10.3389/fphys.2016.00580

    Figure Lengend Snippet: The immunoblots and relative intensities of Prdx6 in membrane-fraction proteins of livers of the fresh water (FW) and seawater (SW) milkfish acclimated to 28°C (white bar) and 18°C (stripe bar) for 1 week, respectively . In the representative immunoblot, single immunoreactive bands at 100 and 25 kDa represent Na + , K + -ATPase (NKA), and Prdx6, respectively. The NKA was used as the loading control of the membrane fraction. Different letters (a and b) indicate significant differences between the 28° and 18°C group, and (x and y) indicate significant differences between the FW and SW group. Values are means ± SEM, n = 6. The Student's t -test pairwise comparison was used following two-way ANOVA, P

    Article Snippet: Then, the immunoblotting was performed as described above except that Na+ , K+ -ATPase (NKA; α5, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) was used as the loading control.

    Techniques: Western Blot