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  • 96
    Thermo Fisher na fluor influenza neuraminidase assay kit
    Measurement of the anti-viral activity of WPS using <t>neuraminidase</t> inhibition and hemagglutination inhibition assays. (A) <t>Influenza</t> A viruses (H1N1 and H3N2) were added at 32 hemagglutination units (HAUs) to the indicated concentrations of WPS, zanamivir (positive control), or PBS (negative control); were mixed with <t>NA-Fluor</t> TM substrate; and were incubated at 37°C for 1 h away from light. Fluorescence was monitored (excitation, 365 nm; emission, 445 nm). Data are representative of two independent experiments, and results were reproducible. (B) Hemagglutination inhibition <t>assay:</t> WPS was serially diluted using PBS and added to equal volumes of the viruses (64 HAUs). To assess red blood cell (RBC) hemolysis inhibition, 50 μL 1% chicken RBCs were added to each well of a 96-well plate and incubated for 1 h at room temperature. Data are representative of two independent experiments, and results were reproducible.
    Na Fluor Influenza Neuraminidase Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore na k atpase
    HCV suppresses expression of ErbB3 and modulates the expression of EGFR and ErbB2 to various extents. For (A) and (B) the hepatoma cell lines Huh7 and Huh7.5 were cultured for 48 hours while primary human hepatocytes (PHH) were isolated and cultured for 24 hours. Subsequently (A) total mRNA was analysed for transcript abundance of EGFR, ErbB2, ErbB3 and ErbB4 by rtPCR and for (B) total protein lysates were prepared and protein expression of EGFR, ErbB2 and ErbB3 was analysed by immunoblot using specific antibodies. β-actin or GAPDH levels were determined as loading controls. For (C) to (E) Huh cell lines harbouring the subgenomic replicon of HCV genotype 1b and the respective control cell line Huh7 cells were cultured for 48 hours and thereafter for (C) total RNA and for (D and E) total protein extracts were prepared and analysed for the abundance of EGFR, ErbB2, ErbB3 and ErbB4 transcripts (C) or for the protein levels of ErbB3 (D) as well as EGFR and ErbB2 (E). Additionally, NS3 expression was assessed for control of replication and β-actin levels for loading control. For (F) Huh9-13 cells were cultured for 24 hours and subsequently treated with 2-C’-Methylcytidine as indicated. After an additional 48 hours total protein extracts were prepared and analysed for ErbB3 and NS3 protein levels by immunoblot. (G and H) Huh7.5 cells were infected with 1 MOI of the HCVcc strain JC1 or left un-infected for control. Total RNA or protein lysates were prepared 48 hours after infection for quantification of the ErbB3 transcript (G) or 72 hours after infection for abundance of the respective protein by immunoblot using antibodies specifically recognizing ErbB3, NS5A or β-actin (H). For (I) Huh7.5 cells were differentiated by DMSO as summarized in the Material and Methods section and infected with the JC1 virus or left uninfected for control. Four weeks after infection total protein extracts were prepared and ErbB3 protein abundance was determined. To prove the differentiation status of the respective cell models used, Huh7.5 cells were either cultured for 5 days without DMSO treatment (H) or for 4 weeks in the presence of DMSO (I). After the respective culture period cells were fixed with ice cold methanol and analysed by immunofluorescence and confocal laser scanning imaging for the Na + /K + <t>ATPase</t> as a basolateral marker (red) and for the presence of MRP2 which indicates polarization and formation of an apical compartment (green). Since MRP2 is only expressed in the apical membrane positive staining for MRP2 indicates cellular differentiation coinciding with polarization (compare immunofluorescence depicted in (H) with that in (I)). For (A), (C) and (G) semiquantitative rtPCR results were calculated using the ΔΔCT method and SDHA as control gene and for (A) data are presented as means + SEM of at least seven or more independent experiments. For (C) and (G) data are provided as fractions of the respective control cells which were set to one and are depicted as means + SEM of at least three independent experiments. The blots presented in (D) to (F), (H) and (I) were evaluated densitometrically and relative expression of ErbB3 (D,F,H and I), EGFR or ErbB2 (E) was normalized to β-actin and data are expressed as fractions of the normalized value of the respective control, which was set to one. Data are presented as means + SEM of at least three or more independent experiments. p ≤ 0.05 was considered to be significant.
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    R&D Systems mouse il 1 beta il 1f2 antibody
    HCV suppresses expression of ErbB3 and modulates the expression of EGFR and ErbB2 to various extents. For (A) and (B) the hepatoma cell lines Huh7 and Huh7.5 were cultured for 48 hours while primary human hepatocytes (PHH) were isolated and cultured for 24 hours. Subsequently (A) total mRNA was analysed for transcript abundance of EGFR, ErbB2, ErbB3 and ErbB4 by rtPCR and for (B) total protein lysates were prepared and protein expression of EGFR, ErbB2 and ErbB3 was analysed by immunoblot using specific antibodies. β-actin or GAPDH levels were determined as loading controls. For (C) to (E) Huh cell lines harbouring the subgenomic replicon of HCV genotype 1b and the respective control cell line Huh7 cells were cultured for 48 hours and thereafter for (C) total RNA and for (D and E) total protein extracts were prepared and analysed for the abundance of EGFR, ErbB2, ErbB3 and ErbB4 transcripts (C) or for the protein levels of ErbB3 (D) as well as EGFR and ErbB2 (E). Additionally, NS3 expression was assessed for control of replication and β-actin levels for loading control. For (F) Huh9-13 cells were cultured for 24 hours and subsequently treated with 2-C’-Methylcytidine as indicated. After an additional 48 hours total protein extracts were prepared and analysed for ErbB3 and NS3 protein levels by immunoblot. (G and H) Huh7.5 cells were infected with 1 MOI of the HCVcc strain JC1 or left un-infected for control. Total RNA or protein lysates were prepared 48 hours after infection for quantification of the ErbB3 transcript (G) or 72 hours after infection for abundance of the respective protein by immunoblot using antibodies specifically recognizing ErbB3, NS5A or β-actin (H). For (I) Huh7.5 cells were differentiated by DMSO as summarized in the Material and Methods section and infected with the JC1 virus or left uninfected for control. Four weeks after infection total protein extracts were prepared and ErbB3 protein abundance was determined. To prove the differentiation status of the respective cell models used, Huh7.5 cells were either cultured for 5 days without DMSO treatment (H) or for 4 weeks in the presence of DMSO (I). After the respective culture period cells were fixed with ice cold methanol and analysed by immunofluorescence and confocal laser scanning imaging for the Na + /K + <t>ATPase</t> as a basolateral marker (red) and for the presence of MRP2 which indicates polarization and formation of an apical compartment (green). Since MRP2 is only expressed in the apical membrane positive staining for MRP2 indicates cellular differentiation coinciding with polarization (compare immunofluorescence depicted in (H) with that in (I)). For (A), (C) and (G) semiquantitative rtPCR results were calculated using the ΔΔCT method and SDHA as control gene and for (A) data are presented as means + SEM of at least seven or more independent experiments. For (C) and (G) data are provided as fractions of the respective control cells which were set to one and are depicted as means + SEM of at least three independent experiments. The blots presented in (D) to (F), (H) and (I) were evaluated densitometrically and relative expression of ErbB3 (D,F,H and I), EGFR or ErbB2 (E) was normalized to β-actin and data are expressed as fractions of the normalized value of the respective control, which was set to one. Data are presented as means + SEM of at least three or more independent experiments. p ≤ 0.05 was considered to be significant.
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    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
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    91
    Vollrath bermingham na
    Hair cell generation through lateral inhibition in the crista A) In cristae explanted from P7 and P30 mice cultured for 5 DIV, Hes5-eGFP is strongly expressed in the peripheral support cells. Upon Notch inhibition with DAPT, Hes5 is downregulated. B) The cre recombinase/reporter strategy used to lineage trace support cells in the mature cristae. Mice expressing PLP-creER, which is expressed only in peripheral support cells of the cristae, were crossed with R26-mTmG mice. Upon Tamoxifen treatment, PLP-expressing cells began expressing membrane-bound GFP (mGFP). C) An example of a lineage traced transitional cell (green) expressing the early hair cell marker Gfi1 (arrow) and possessing a normal hair cell morphology, including a kinocilium. [Reprinted with kind permission from Springer Science + Business Media: Slowik, A. D. and <t>Bermingham-McDonogh,</t> O. 2013, J. Assoc. Res. Otolaryngol., 14, 813.]
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    92
    Abcam na k atpase
    Micrographs of kidney biopsy serial sections ( A , B ) and transmission electron micrographs of kidney biopsy sections ( C – F ) from patients with Fabry nephropathy. PAS, elastica Masson trichrome, anti-UMOD Ab, anti-NKCC2 Ab, anti-Na + -K + <t>-ATPase</t> Ab, and anti-AQP2 Ab staining. A ) Inner stripe: upper row, control [Cont (1)] lower row, Fabry nephropathy [Fabry (B)]. B , respectively. Scale bars, 20 μm. C ) mTAL of patient A. D ) Cortical TAL (cTAL) of patient I. E ) CD of patient A. F ) Arteriole of patient A. Arrows indicate basolateral infolding. Scale bars, 10 μm. AVR, ascending vasa recta; E, endothelial cell; F, fibrosis; G, glomerulus; ICC, intercalated cell; M, mitochondria; S, sloughed lamellar body; SM, smooth muscle cell.
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    Santa Cruz Biotechnology na k atpase
    Expression of <t>Na+/K+</t> <t>ATPase.</t> Cell lysates obtained from A549 cells treated with PBS (mock), TNF-α (10 ng/ml), two dosages of recombinant B19-VP1u and HBoV-VP1u (400 ng/ml and 4000 ng/ml) and bee venom PLA2 (10 ng/ml) were probed with antibodies against Na+/K+ ATPase. Quantified result was shown in the lower panel. Similar results were observed in three independent experiments and * indicates the significant difference as compared to the mock, P
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    92
    Olympus na water immersion objective
    Expression of <t>Na+/K+</t> <t>ATPase.</t> Cell lysates obtained from A549 cells treated with PBS (mock), TNF-α (10 ng/ml), two dosages of recombinant B19-VP1u and HBoV-VP1u (400 ng/ml and 4000 ng/ml) and bee venom PLA2 (10 ng/ml) were probed with antibodies against Na+/K+ ATPase. Quantified result was shown in the lower panel. Similar results were observed in three independent experiments and * indicates the significant difference as compared to the mock, P
    Na Water Immersion Objective, supplied by Olympus, used in various techniques. Bioz Stars score: 92/100, based on 475 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc na k atpase
    Ser163 modulates interaction with <t>Na,K-ATPase.</t> Immune complexes were precipitated from crude membrane preparations of HEK 293 cells using antibodies against the α-subunit of the Na,K-ATPase (IP:α 1, 3A), against the M2-Flag epitope (IP:Flag,
    Na K Atpase, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 93/100, based on 237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Carl Zeiss na plan apochromat objective
    Ser163 modulates interaction with <t>Na,K-ATPase.</t> Immune complexes were precipitated from crude membrane preparations of HEK 293 cells using antibodies against the α-subunit of the Na,K-ATPase (IP:α 1, 3A), against the M2-Flag epitope (IP:Flag,
    Na Plan Apochromat Objective, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 91/100, based on 596 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    R&D Systems caspase 3
    Detection of activated <t>caspase</t> 3 as a marker for cells undergoing apoptosis in psoriatic patients before and after treatment with adalimumab
    Caspase 3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 1282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Carl Zeiss na water immersion objective
    Detection of activated <t>caspase</t> 3 as a marker for cells undergoing apoptosis in psoriatic patients before and after treatment with adalimumab
    Na Water Immersion Objective, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 92/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Olympus na oil immersion objective
    Signal attenuation of 0.2 μm fluorescent microspheres mounted in kidney tissue. Images were collected with a 60×oil immersion objective NA 1.4 with immersion oil (refractive index 1.515) or a 60× water immersion objective NA1.2
    Na Oil Immersion Objective, supplied by Olympus, used in various techniques. Bioz Stars score: 92/100, based on 470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Developmental Studies Hybridoma Bank na k atpase
    Pronephric morphogenesis, patterning, and segmentation in control and morphant embryos. (A–D) Expression of pax2.1 in 1-dpf embryos or cdh17 in 2-dpf embryos as revealed by in situ hybridization. (E, F) Low magnification overview of Na + /K + <t>-ATPase</t> expression in the PTs of 2-dpf control or morphant embryos. (G–P) Expression of wt1a (G–H), nph2s (I, M), slc4a4 (J,N), slc12a (K, O) or clck (L, P) in control or morphant 3-dpf embryos.
    Na K Atpase, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 93/100, based on 318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti na k atpase
    TMEM30a-GFP localizes in the plasma membrane and internal organelles (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask ™ Orange Plasma Membrane to mark the plasma membrane ( top ) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum ( row 2 ), or Golgi ( row 3 ). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria ( bottom ). (B) Western blot for GFP or plasma membrane <t>Na/K</t> <t>ATPase</t> in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.
    Anti Na K Atpase, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Upchurch Scientific dewyer na
    TMEM30a-GFP localizes in the plasma membrane and internal organelles (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask ™ Orange Plasma Membrane to mark the plasma membrane ( top ) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum ( row 2 ), or Golgi ( row 3 ). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria ( bottom ). (B) Western blot for GFP or plasma membrane <t>Na/K</t> <t>ATPase</t> in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.
    Dewyer Na, supplied by Upchurch Scientific, used in various techniques. Bioz Stars score: 89/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human vegf165 antibody
    TMEM30a-GFP localizes in the plasma membrane and internal organelles (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask ™ Orange Plasma Membrane to mark the plasma membrane ( top ) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum ( row 2 ), or Golgi ( row 3 ). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria ( bottom ). (B) Western blot for GFP or plasma membrane <t>Na/K</t> <t>ATPase</t> in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.
    Human Vegf165 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore na deoxycholate
    TMEM30a-GFP localizes in the plasma membrane and internal organelles (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask ™ Orange Plasma Membrane to mark the plasma membrane ( top ) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum ( row 2 ), or Golgi ( row 3 ). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria ( bottom ). (B) Western blot for GFP or plasma membrane <t>Na/K</t> <t>ATPase</t> in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.
    Na Deoxycholate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1754 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems tgf beta pan specific antibody
    Schematic representation summarizing the transforming growth <t>factors-beta</t> <t>(TGF-β)</t> receptor complexes and signaling pathway in endothelial cells. The green arrowhead lines represent positive crosstalk interactions and steps. Red flat-ended lines indicate inhibition. In the nucleus, the green arrowhead and red flat-ended lines on the DNA represent activation and inhibition of gene expression, respectively. ( a ) LAP-TGF-β latent complex. ( b ) Release of TGF-β from the complex with LAP. ( c ) TGF-β binding to the heterotetrameric receptor complex. ( d ) TβRII phosphorylation of type I receptor ALK5. ( e ) SMAD2/3 phosphorylation by ALK5. ( f ) SMAD1/5/8 phosphorylation by ALK1. ( g ) Dimerization of R-SMADs with SMAD4. ( h ) Translocation of the R-SMAD/SMAD4 complex into the nucleus and binding to regulatory sequences. See text for details.
    Tgf Beta Pan Specific Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse tnf alpha antibody
    Schematic representation summarizing the transforming growth <t>factors-beta</t> <t>(TGF-β)</t> receptor complexes and signaling pathway in endothelial cells. The green arrowhead lines represent positive crosstalk interactions and steps. Red flat-ended lines indicate inhibition. In the nucleus, the green arrowhead and red flat-ended lines on the DNA represent activation and inhibition of gene expression, respectively. ( a ) LAP-TGF-β latent complex. ( b ) Release of TGF-β from the complex with LAP. ( c ) TGF-β binding to the heterotetrameric receptor complex. ( d ) TβRII phosphorylation of type I receptor ALK5. ( e ) SMAD2/3 phosphorylation by ALK5. ( f ) SMAD1/5/8 phosphorylation by ALK1. ( g ) Dimerization of R-SMADs with SMAD4. ( h ) Translocation of the R-SMAD/SMAD4 complex into the nucleus and binding to regulatory sequences. See text for details.
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    R&D Systems human il 6 antibody
    <t>IL-6</t> and BMP-6 induces AR activation in LNCaP cells (a) HTS33B and HPS33Q human prostate stromal cells were cultured in RPMI1640 supplemented with 0.2% charcoal-stripped FBS and treated with TGF-β1 (50 pM) for 3 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of IL-6 and BMP-6. Data is from three independent experiments. (b) LNCaP cells were co-cultured with HTS33B cells in RPMI1640 supplemented with 0.2% charcoal-stripped FBS, and treated with TGF-β1 (50 pM), IL-6 (25 ng/ml), and/or BMP-6 (50 ng/ml) for 6 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of PSA, KLK4, TMPRSS2 expresssion in these co-cultures. Data is from three independent experiments. (c) LNCaP cells were similarly co-cultured with HPS33Q cells and treated with TGF-β1 (50 pM), IL-6 (25 ng/ml), and/or BMP-6 (50 ng/ml) for 6 days, and analyzed for the expression of PSA, KLK4, TMPRSS2 (qPCR). Data is from three independent experiments. All gene expression data were normalized to GAPDH expression. hPS (human prostate stromal cells) stands for either HTS33B (b) or HPS33Q cells (c) . *p
    Human Il 6 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore na phosphate buffer
    <t>IL-6</t> and BMP-6 induces AR activation in LNCaP cells (a) HTS33B and HPS33Q human prostate stromal cells were cultured in RPMI1640 supplemented with 0.2% charcoal-stripped FBS and treated with TGF-β1 (50 pM) for 3 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of IL-6 and BMP-6. Data is from three independent experiments. (b) LNCaP cells were co-cultured with HTS33B cells in RPMI1640 supplemented with 0.2% charcoal-stripped FBS, and treated with TGF-β1 (50 pM), IL-6 (25 ng/ml), and/or BMP-6 (50 ng/ml) for 6 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of PSA, KLK4, TMPRSS2 expresssion in these co-cultures. Data is from three independent experiments. (c) LNCaP cells were similarly co-cultured with HPS33Q cells and treated with TGF-β1 (50 pM), IL-6 (25 ng/ml), and/or BMP-6 (50 ng/ml) for 6 days, and analyzed for the expression of PSA, KLK4, TMPRSS2 (qPCR). Data is from three independent experiments. All gene expression data were normalized to GAPDH expression. hPS (human prostate stromal cells) stands for either HTS33B (b) or HPS33Q cells (c) . *p
    Na Phosphate Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Activated neutrophils are more sensitive to NK cell cytotoxicity compared to resting neutrophils. (A) Percentage of dead neutrophils (PMN) after a 3 h co-culture of neutrophils [pre-stimulated in vitro with <t>CL097</t> and <t>GM-CSF,</t> or kept on ice (ctrl)] and bulk-NK cells at an E:T ratio of 10:1, with addition of an HLA antibody as indicated ( n = 7; one-way ANOVA followed by Dunnett's multiple comparisons test). (B) Graph shows percentage of dead neutrophils after a 4 h cytotoxicity assay performed using autologous NK effector cells toward transmigrated (ePMN) or blood neutrophils (bPMN; E:T ratio 10:1; n = 5; one-way ANOVA followed by Dunnett's multiple comparisons test). Error bars represent SEM. (C) Representative FACS plots showing percentage of dead neutrophils (transmigrated or resting) measured using AnnexinV and To-Pro-3, after a 4 h co-culture with autologous NK cells.
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    Activated neutrophils are more sensitive to NK cell cytotoxicity compared to resting neutrophils. (A) Percentage of dead neutrophils (PMN) after a 3 h co-culture of neutrophils [pre-stimulated in vitro with <t>CL097</t> and <t>GM-CSF,</t> or kept on ice (ctrl)] and bulk-NK cells at an E:T ratio of 10:1, with addition of an HLA antibody as indicated ( n = 7; one-way ANOVA followed by Dunnett's multiple comparisons test). (B) Graph shows percentage of dead neutrophils after a 4 h cytotoxicity assay performed using autologous NK effector cells toward transmigrated (ePMN) or blood neutrophils (bPMN; E:T ratio 10:1; n = 5; one-way ANOVA followed by Dunnett's multiple comparisons test). Error bars represent SEM. (C) Representative FACS plots showing percentage of dead neutrophils (transmigrated or resting) measured using AnnexinV and To-Pro-3, after a 4 h co-culture with autologous NK cells.
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    Image Search Results


    Measurement of the anti-viral activity of WPS using neuraminidase inhibition and hemagglutination inhibition assays. (A) Influenza A viruses (H1N1 and H3N2) were added at 32 hemagglutination units (HAUs) to the indicated concentrations of WPS, zanamivir (positive control), or PBS (negative control); were mixed with NA-Fluor TM substrate; and were incubated at 37°C for 1 h away from light. Fluorescence was monitored (excitation, 365 nm; emission, 445 nm). Data are representative of two independent experiments, and results were reproducible. (B) Hemagglutination inhibition assay: WPS was serially diluted using PBS and added to equal volumes of the viruses (64 HAUs). To assess red blood cell (RBC) hemolysis inhibition, 50 μL 1% chicken RBCs were added to each well of a 96-well plate and incubated for 1 h at room temperature. Data are representative of two independent experiments, and results were reproducible.

    Journal: Frontiers in Pharmacology

    Article Title: In vitro Anti-viral Activity of Psoraleae Semen Water Extract against Influenza A Viruses

    doi: 10.3389/fphar.2016.00460

    Figure Lengend Snippet: Measurement of the anti-viral activity of WPS using neuraminidase inhibition and hemagglutination inhibition assays. (A) Influenza A viruses (H1N1 and H3N2) were added at 32 hemagglutination units (HAUs) to the indicated concentrations of WPS, zanamivir (positive control), or PBS (negative control); were mixed with NA-Fluor TM substrate; and were incubated at 37°C for 1 h away from light. Fluorescence was monitored (excitation, 365 nm; emission, 445 nm). Data are representative of two independent experiments, and results were reproducible. (B) Hemagglutination inhibition assay: WPS was serially diluted using PBS and added to equal volumes of the viruses (64 HAUs). To assess red blood cell (RBC) hemolysis inhibition, 50 μL 1% chicken RBCs were added to each well of a 96-well plate and incubated for 1 h at room temperature. Data are representative of two independent experiments, and results were reproducible.

    Article Snippet: The NA inhibition assay was performed according to the manufacturer’s instructions using the NA-Fluor Influenza Neuraminidase Assay kit (Life Technologies, Carlsbad, CA, USA).

    Techniques: Activity Assay, Inhibition, HI Assay, Positive Control, Negative Control, Incubation, Fluorescence

    HCV suppresses expression of ErbB3 and modulates the expression of EGFR and ErbB2 to various extents. For (A) and (B) the hepatoma cell lines Huh7 and Huh7.5 were cultured for 48 hours while primary human hepatocytes (PHH) were isolated and cultured for 24 hours. Subsequently (A) total mRNA was analysed for transcript abundance of EGFR, ErbB2, ErbB3 and ErbB4 by rtPCR and for (B) total protein lysates were prepared and protein expression of EGFR, ErbB2 and ErbB3 was analysed by immunoblot using specific antibodies. β-actin or GAPDH levels were determined as loading controls. For (C) to (E) Huh cell lines harbouring the subgenomic replicon of HCV genotype 1b and the respective control cell line Huh7 cells were cultured for 48 hours and thereafter for (C) total RNA and for (D and E) total protein extracts were prepared and analysed for the abundance of EGFR, ErbB2, ErbB3 and ErbB4 transcripts (C) or for the protein levels of ErbB3 (D) as well as EGFR and ErbB2 (E). Additionally, NS3 expression was assessed for control of replication and β-actin levels for loading control. For (F) Huh9-13 cells were cultured for 24 hours and subsequently treated with 2-C’-Methylcytidine as indicated. After an additional 48 hours total protein extracts were prepared and analysed for ErbB3 and NS3 protein levels by immunoblot. (G and H) Huh7.5 cells were infected with 1 MOI of the HCVcc strain JC1 or left un-infected for control. Total RNA or protein lysates were prepared 48 hours after infection for quantification of the ErbB3 transcript (G) or 72 hours after infection for abundance of the respective protein by immunoblot using antibodies specifically recognizing ErbB3, NS5A or β-actin (H). For (I) Huh7.5 cells were differentiated by DMSO as summarized in the Material and Methods section and infected with the JC1 virus or left uninfected for control. Four weeks after infection total protein extracts were prepared and ErbB3 protein abundance was determined. To prove the differentiation status of the respective cell models used, Huh7.5 cells were either cultured for 5 days without DMSO treatment (H) or for 4 weeks in the presence of DMSO (I). After the respective culture period cells were fixed with ice cold methanol and analysed by immunofluorescence and confocal laser scanning imaging for the Na + /K + ATPase as a basolateral marker (red) and for the presence of MRP2 which indicates polarization and formation of an apical compartment (green). Since MRP2 is only expressed in the apical membrane positive staining for MRP2 indicates cellular differentiation coinciding with polarization (compare immunofluorescence depicted in (H) with that in (I)). For (A), (C) and (G) semiquantitative rtPCR results were calculated using the ΔΔCT method and SDHA as control gene and for (A) data are presented as means + SEM of at least seven or more independent experiments. For (C) and (G) data are provided as fractions of the respective control cells which were set to one and are depicted as means + SEM of at least three independent experiments. The blots presented in (D) to (F), (H) and (I) were evaluated densitometrically and relative expression of ErbB3 (D,F,H and I), EGFR or ErbB2 (E) was normalized to β-actin and data are expressed as fractions of the normalized value of the respective control, which was set to one. Data are presented as means + SEM of at least three or more independent experiments. p ≤ 0.05 was considered to be significant.

    Journal: PLoS ONE

    Article Title: Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family

    doi: 10.1371/journal.pone.0148711

    Figure Lengend Snippet: HCV suppresses expression of ErbB3 and modulates the expression of EGFR and ErbB2 to various extents. For (A) and (B) the hepatoma cell lines Huh7 and Huh7.5 were cultured for 48 hours while primary human hepatocytes (PHH) were isolated and cultured for 24 hours. Subsequently (A) total mRNA was analysed for transcript abundance of EGFR, ErbB2, ErbB3 and ErbB4 by rtPCR and for (B) total protein lysates were prepared and protein expression of EGFR, ErbB2 and ErbB3 was analysed by immunoblot using specific antibodies. β-actin or GAPDH levels were determined as loading controls. For (C) to (E) Huh cell lines harbouring the subgenomic replicon of HCV genotype 1b and the respective control cell line Huh7 cells were cultured for 48 hours and thereafter for (C) total RNA and for (D and E) total protein extracts were prepared and analysed for the abundance of EGFR, ErbB2, ErbB3 and ErbB4 transcripts (C) or for the protein levels of ErbB3 (D) as well as EGFR and ErbB2 (E). Additionally, NS3 expression was assessed for control of replication and β-actin levels for loading control. For (F) Huh9-13 cells were cultured for 24 hours and subsequently treated with 2-C’-Methylcytidine as indicated. After an additional 48 hours total protein extracts were prepared and analysed for ErbB3 and NS3 protein levels by immunoblot. (G and H) Huh7.5 cells were infected with 1 MOI of the HCVcc strain JC1 or left un-infected for control. Total RNA or protein lysates were prepared 48 hours after infection for quantification of the ErbB3 transcript (G) or 72 hours after infection for abundance of the respective protein by immunoblot using antibodies specifically recognizing ErbB3, NS5A or β-actin (H). For (I) Huh7.5 cells were differentiated by DMSO as summarized in the Material and Methods section and infected with the JC1 virus or left uninfected for control. Four weeks after infection total protein extracts were prepared and ErbB3 protein abundance was determined. To prove the differentiation status of the respective cell models used, Huh7.5 cells were either cultured for 5 days without DMSO treatment (H) or for 4 weeks in the presence of DMSO (I). After the respective culture period cells were fixed with ice cold methanol and analysed by immunofluorescence and confocal laser scanning imaging for the Na + /K + ATPase as a basolateral marker (red) and for the presence of MRP2 which indicates polarization and formation of an apical compartment (green). Since MRP2 is only expressed in the apical membrane positive staining for MRP2 indicates cellular differentiation coinciding with polarization (compare immunofluorescence depicted in (H) with that in (I)). For (A), (C) and (G) semiquantitative rtPCR results were calculated using the ΔΔCT method and SDHA as control gene and for (A) data are presented as means + SEM of at least seven or more independent experiments. For (C) and (G) data are provided as fractions of the respective control cells which were set to one and are depicted as means + SEM of at least three independent experiments. The blots presented in (D) to (F), (H) and (I) were evaluated densitometrically and relative expression of ErbB3 (D,F,H and I), EGFR or ErbB2 (E) was normalized to β-actin and data are expressed as fractions of the normalized value of the respective control, which was set to one. Data are presented as means + SEM of at least three or more independent experiments. p ≤ 0.05 was considered to be significant.

    Article Snippet: MRP2 (#MA1-26536) was purchased from Thermo Fisher Scientific (Waltham, USA), Na+ /K+ ATPase (#A276) from Sigma Aldrich (St. Louis, USA).

    Techniques: Expressing, Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Infection, Western Blot, Immunofluorescence, Imaging, Marker, Staining, Cell Differentiation

    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

    Hair cell generation through lateral inhibition in the crista A) In cristae explanted from P7 and P30 mice cultured for 5 DIV, Hes5-eGFP is strongly expressed in the peripheral support cells. Upon Notch inhibition with DAPT, Hes5 is downregulated. B) The cre recombinase/reporter strategy used to lineage trace support cells in the mature cristae. Mice expressing PLP-creER, which is expressed only in peripheral support cells of the cristae, were crossed with R26-mTmG mice. Upon Tamoxifen treatment, PLP-expressing cells began expressing membrane-bound GFP (mGFP). C) An example of a lineage traced transitional cell (green) expressing the early hair cell marker Gfi1 (arrow) and possessing a normal hair cell morphology, including a kinocilium. [Reprinted with kind permission from Springer Science + Business Media: Slowik, A. D. and Bermingham-McDonogh, O. 2013, J. Assoc. Res. Otolaryngol., 14, 813.]

    Journal: Trends in developmental biology

    Article Title: Notch signaling in mammalian hair cell regeneration

    doi:

    Figure Lengend Snippet: Hair cell generation through lateral inhibition in the crista A) In cristae explanted from P7 and P30 mice cultured for 5 DIV, Hes5-eGFP is strongly expressed in the peripheral support cells. Upon Notch inhibition with DAPT, Hes5 is downregulated. B) The cre recombinase/reporter strategy used to lineage trace support cells in the mature cristae. Mice expressing PLP-creER, which is expressed only in peripheral support cells of the cristae, were crossed with R26-mTmG mice. Upon Tamoxifen treatment, PLP-expressing cells began expressing membrane-bound GFP (mGFP). C) An example of a lineage traced transitional cell (green) expressing the early hair cell marker Gfi1 (arrow) and possessing a normal hair cell morphology, including a kinocilium. [Reprinted with kind permission from Springer Science + Business Media: Slowik, A. D. and Bermingham-McDonogh, O. 2013, J. Assoc. Res. Otolaryngol., 14, 813.]

    Article Snippet: Bermingham NA, Hassan BA, Price SD, Vollrath MA, Ben-Arie N, Eatock RA, Bellen HJ, Lysakowski A, Zoghbi HY.

    Techniques: Inhibition, Mouse Assay, Cell Culture, Expressing, Plasmid Purification, Marker

    Micrographs of kidney biopsy serial sections ( A , B ) and transmission electron micrographs of kidney biopsy sections ( C – F ) from patients with Fabry nephropathy. PAS, elastica Masson trichrome, anti-UMOD Ab, anti-NKCC2 Ab, anti-Na + -K + -ATPase Ab, and anti-AQP2 Ab staining. A ) Inner stripe: upper row, control [Cont (1)] lower row, Fabry nephropathy [Fabry (B)]. B , respectively. Scale bars, 20 μm. C ) mTAL of patient A. D ) Cortical TAL (cTAL) of patient I. E ) CD of patient A. F ) Arteriole of patient A. Arrows indicate basolateral infolding. Scale bars, 10 μm. AVR, ascending vasa recta; E, endothelial cell; F, fibrosis; G, glomerulus; ICC, intercalated cell; M, mitochondria; S, sloughed lamellar body; SM, smooth muscle cell.

    Journal: The FASEB Journal

    Article Title: Medullary thick ascending limb impairment in the GlatmTg(CAG-A4GALT) Fabry model mice

    doi: 10.1096/fj.201701374R

    Figure Lengend Snippet: Micrographs of kidney biopsy serial sections ( A , B ) and transmission electron micrographs of kidney biopsy sections ( C – F ) from patients with Fabry nephropathy. PAS, elastica Masson trichrome, anti-UMOD Ab, anti-NKCC2 Ab, anti-Na + -K + -ATPase Ab, and anti-AQP2 Ab staining. A ) Inner stripe: upper row, control [Cont (1)] lower row, Fabry nephropathy [Fabry (B)]. B , respectively. Scale bars, 20 μm. C ) mTAL of patient A. D ) Cortical TAL (cTAL) of patient I. E ) CD of patient A. F ) Arteriole of patient A. Arrows indicate basolateral infolding. Scale bars, 10 μm. AVR, ascending vasa recta; E, endothelial cell; F, fibrosis; G, glomerulus; ICC, intercalated cell; M, mitochondria; S, sloughed lamellar body; SM, smooth muscle cell.

    Article Snippet: UMOD interacts with NKCC2 in apical trafficking ( , ) and regulates the turnover, trafficking, and basolateral expression of Na+ -K+ -ATPase ( ).

    Techniques: Transmission Assay, Staining, Immunocytochemistry

    Gla tm Tg(CAG-A4GALT) mice demonstrate reduced expression of ion transport–related molecules in the TAL. A ) Representative transmission electron micrographs of the mTAL in 20-wk-old Gla tm Tg(CAG-A4GALT) and WT mice ( n = 2/group). Arrows indicate basolateral infolding, and arrowhead indicates a lamellar body. Scale bars, 1 μm. B ) Representative images of UMOD immunoreactivity in kidneys of Gla tm Tg(CAG-A4GALT) and WT mice ( n = 3/group). Scale bars, 1 mm. C ) Micrographs of UMOD expression in TAL of Gla tm Tg(CAG-A4GALT) and WT mice. Arrows indicate a vacuolated TAL cell. Scale bars, 10 μm. D ) Representative images of NKCC2 immunoreactivity in kidneys of Gla tm Tg(CAG-A4GALT) and WT mice ( n = 3/group). Scale bars, 1 mm. E ) Micrographs of NKCC2 expression in TAL of Gla tm Tg(CAG-A4GALT) and WT mice. Arrows indicate TAL cells with weak NKCC2 staining. Scale bars, 10 μm. F ) Representative images of Na + -K + -ATPase immunoreactivity in kidneys of Gla tm Tg(CAG-A4GALT) and WT mice ( n = 3/group). Scale bars, 1 mm. G ) Micrographs of Na + -K + -ATPase expression in TAL of Gla tm Tg(CAG-A4GALT) and WT mice. Arrows indicate TAL cells with weak Na + -K + -ATPase staining. Scale bars, 10 μm. BM, basement membrane; G, glomerulus; M, mitochondria; MD, macula densa.

    Journal: The FASEB Journal

    Article Title: Medullary thick ascending limb impairment in the GlatmTg(CAG-A4GALT) Fabry model mice

    doi: 10.1096/fj.201701374R

    Figure Lengend Snippet: Gla tm Tg(CAG-A4GALT) mice demonstrate reduced expression of ion transport–related molecules in the TAL. A ) Representative transmission electron micrographs of the mTAL in 20-wk-old Gla tm Tg(CAG-A4GALT) and WT mice ( n = 2/group). Arrows indicate basolateral infolding, and arrowhead indicates a lamellar body. Scale bars, 1 μm. B ) Representative images of UMOD immunoreactivity in kidneys of Gla tm Tg(CAG-A4GALT) and WT mice ( n = 3/group). Scale bars, 1 mm. C ) Micrographs of UMOD expression in TAL of Gla tm Tg(CAG-A4GALT) and WT mice. Arrows indicate a vacuolated TAL cell. Scale bars, 10 μm. D ) Representative images of NKCC2 immunoreactivity in kidneys of Gla tm Tg(CAG-A4GALT) and WT mice ( n = 3/group). Scale bars, 1 mm. E ) Micrographs of NKCC2 expression in TAL of Gla tm Tg(CAG-A4GALT) and WT mice. Arrows indicate TAL cells with weak NKCC2 staining. Scale bars, 10 μm. F ) Representative images of Na + -K + -ATPase immunoreactivity in kidneys of Gla tm Tg(CAG-A4GALT) and WT mice ( n = 3/group). Scale bars, 1 mm. G ) Micrographs of Na + -K + -ATPase expression in TAL of Gla tm Tg(CAG-A4GALT) and WT mice. Arrows indicate TAL cells with weak Na + -K + -ATPase staining. Scale bars, 10 μm. BM, basement membrane; G, glomerulus; M, mitochondria; MD, macula densa.

    Article Snippet: UMOD interacts with NKCC2 in apical trafficking ( , ) and regulates the turnover, trafficking, and basolateral expression of Na+ -K+ -ATPase ( ).

    Techniques: Mouse Assay, Expressing, Transmission Assay, Staining

    Gla tm Tg(CAG-A4GALT) mice demonstrate reduced expression of Umod (UMOD), Slc12a1 (NKCC2), and Na + -K + -ATPase in the whole kidney. A ) Real-time RT-PCR analysis of Umod mRNA levels in Gla tm Tg(CAG-A4GALT) and WT mice. Gla tm Tg(CAG-A4GALT) mice: 5 ( n = 7), 10 ( n = 8), and 20 wk old ( n = 7); WT mice: 5 ( n = 7), 10 ( n = 8), and 20 wk old ( n = 7). B ) Representative (of 2 experiments) Western blot analysis of UMOD expression in Gla tm Tg(CAG-A4GALT) ( n = 7) and WT mice ( n = 5). C ) Real-time RT-PCR analyses for Slc12a1 expression levels in the same mice as in panel A . D ) Representative (of 2 experiments) Western blot analysis of NKCC2 expression in the same mice as in panel B . E ) Representative (of 2 experiments) Western blot analysis of Na + -K + -ATPase expression in Gla tm Tg(CAG-A4GALT) ( n = 6) and WT mice ( n = 6). In box-and-whisker plots, center lines represent the median, limits represent quartiles, whiskers represent the 10th and 90th percentiles, and red lines represent the mean. Differences between groups were evaluated by using Student’s t test; data are shown as t (integral degree of freedom) = t , P . For Welch’s t test, data are shown as t (mixed decimal degree of freedom) = t , P . For the Wilcoxon rank-sum test, data are shown with a P value only.

    Journal: The FASEB Journal

    Article Title: Medullary thick ascending limb impairment in the GlatmTg(CAG-A4GALT) Fabry model mice

    doi: 10.1096/fj.201701374R

    Figure Lengend Snippet: Gla tm Tg(CAG-A4GALT) mice demonstrate reduced expression of Umod (UMOD), Slc12a1 (NKCC2), and Na + -K + -ATPase in the whole kidney. A ) Real-time RT-PCR analysis of Umod mRNA levels in Gla tm Tg(CAG-A4GALT) and WT mice. Gla tm Tg(CAG-A4GALT) mice: 5 ( n = 7), 10 ( n = 8), and 20 wk old ( n = 7); WT mice: 5 ( n = 7), 10 ( n = 8), and 20 wk old ( n = 7). B ) Representative (of 2 experiments) Western blot analysis of UMOD expression in Gla tm Tg(CAG-A4GALT) ( n = 7) and WT mice ( n = 5). C ) Real-time RT-PCR analyses for Slc12a1 expression levels in the same mice as in panel A . D ) Representative (of 2 experiments) Western blot analysis of NKCC2 expression in the same mice as in panel B . E ) Representative (of 2 experiments) Western blot analysis of Na + -K + -ATPase expression in Gla tm Tg(CAG-A4GALT) ( n = 6) and WT mice ( n = 6). In box-and-whisker plots, center lines represent the median, limits represent quartiles, whiskers represent the 10th and 90th percentiles, and red lines represent the mean. Differences between groups were evaluated by using Student’s t test; data are shown as t (integral degree of freedom) = t , P . For Welch’s t test, data are shown as t (mixed decimal degree of freedom) = t , P . For the Wilcoxon rank-sum test, data are shown with a P value only.

    Article Snippet: UMOD interacts with NKCC2 in apical trafficking ( , ) and regulates the turnover, trafficking, and basolateral expression of Na+ -K+ -ATPase ( ).

    Techniques: Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot, Whisker Assay

    Expression of Na+/K+ ATPase. Cell lysates obtained from A549 cells treated with PBS (mock), TNF-α (10 ng/ml), two dosages of recombinant B19-VP1u and HBoV-VP1u (400 ng/ml and 4000 ng/ml) and bee venom PLA2 (10 ng/ml) were probed with antibodies against Na+/K+ ATPase. Quantified result was shown in the lower panel. Similar results were observed in three independent experiments and * indicates the significant difference as compared to the mock, P

    Journal: PLoS ONE

    Article Title: Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 Cells

    doi: 10.1371/journal.pone.0107970

    Figure Lengend Snippet: Expression of Na+/K+ ATPase. Cell lysates obtained from A549 cells treated with PBS (mock), TNF-α (10 ng/ml), two dosages of recombinant B19-VP1u and HBoV-VP1u (400 ng/ml and 4000 ng/ml) and bee venom PLA2 (10 ng/ml) were probed with antibodies against Na+/K+ ATPase. Quantified result was shown in the lower panel. Similar results were observed in three independent experiments and * indicates the significant difference as compared to the mock, P

    Article Snippet: More significant decreases of Na+/K+ ATPase in A549 cells were detected by treatment with TNF-α, 4000 ug/ml B19-VP1u and both dosages of HBoV-VP1u than that of the controls ( ).

    Techniques: Expressing, Recombinant

    Ser163 modulates interaction with Na,K-ATPase. Immune complexes were precipitated from crude membrane preparations of HEK 293 cells using antibodies against the α-subunit of the Na,K-ATPase (IP:α 1, 3A), against the M2-Flag epitope (IP:Flag,

    Journal:

    Article Title: S163 is critical for FXYD5 modulation of wound healing in airway epithelial cells

    doi: 10.1111/j.1524-475X.2008.00432.x

    Figure Lengend Snippet: Ser163 modulates interaction with Na,K-ATPase. Immune complexes were precipitated from crude membrane preparations of HEK 293 cells using antibodies against the α-subunit of the Na,K-ATPase (IP:α 1, 3A), against the M2-Flag epitope (IP:Flag,

    Article Snippet: Although a primary function of the Na,K-ATPase is to maintain the monovalent cation balance between the interior and the exterior of the cell, it is also a critical participant in establishing polarity of epithelial cells and in cell motility.

    Techniques: FLAG-tag

    FXYD5 expression alters vimentin but not RhoA or Na,K-ATPase expression in LA4 airway cell membrane preparations. (A) Representative GFP fluorescent images of murine LA4 airway cells transfected with pKCEREGFP, demonstrating > 80% transfection

    Journal:

    Article Title: S163 is critical for FXYD5 modulation of wound healing in airway epithelial cells

    doi: 10.1111/j.1524-475X.2008.00432.x

    Figure Lengend Snippet: FXYD5 expression alters vimentin but not RhoA or Na,K-ATPase expression in LA4 airway cell membrane preparations. (A) Representative GFP fluorescent images of murine LA4 airway cells transfected with pKCEREGFP, demonstrating > 80% transfection

    Article Snippet: Although a primary function of the Na,K-ATPase is to maintain the monovalent cation balance between the interior and the exterior of the cell, it is also a critical participant in establishing polarity of epithelial cells and in cell motility.

    Techniques: Expressing, Transfection

    Detection of activated caspase 3 as a marker for cells undergoing apoptosis in psoriatic patients before and after treatment with adalimumab

    Journal:

    Article Title: Targeting TNF? Rapidly Reduces Density of Dendritic Cells and Macrophages in Psoriatic Plaques with Restoration of Epidermal Keratinocyte Differentiation

    doi: 10.1016/j.jdermsci.2007.06.006

    Figure Lengend Snippet: Detection of activated caspase 3 as a marker for cells undergoing apoptosis in psoriatic patients before and after treatment with adalimumab

    Article Snippet: Moreover, there was no detection of a large number of cells expressing activated caspase 3 in any of the treated tissue samples in either epidermal or dermal compartments at days 2, 7, 28, 84, for any of the patients treated with adalimumab ( ).

    Techniques: Marker

    Signal attenuation of 0.2 μm fluorescent microspheres mounted in kidney tissue. Images were collected with a 60×oil immersion objective NA 1.4 with immersion oil (refractive index 1.515) or a 60× water immersion objective NA1.2

    Journal: Journal of microscopy

    Article Title: The effects of spherical aberration on multiphoton fluorescence excitation microscopy

    doi: 10.1111/j.1365-2818.2010.03449.x

    Figure Lengend Snippet: Signal attenuation of 0.2 μm fluorescent microspheres mounted in kidney tissue. Images were collected with a 60×oil immersion objective NA 1.4 with immersion oil (refractive index 1.515) or a 60× water immersion objective NA1.2

    Article Snippet: Once the microsphere was in focus, 300 images were collected at approximately 1 frame per second of this single image plane using an Olympus 60× NA 1.4 oil immersion objective.

    Techniques:

    Fluorescence intensity of 0.2 μm fluorescent microspheres mounted in agarose with varying refractive index, normalized to the average intensity at the surface, versus NFP. Images collected with 60× oil immersion objective NA 1.4 with immersion

    Journal: Journal of microscopy

    Article Title: The effects of spherical aberration on multiphoton fluorescence excitation microscopy

    doi: 10.1111/j.1365-2818.2010.03449.x

    Figure Lengend Snippet: Fluorescence intensity of 0.2 μm fluorescent microspheres mounted in agarose with varying refractive index, normalized to the average intensity at the surface, versus NFP. Images collected with 60× oil immersion objective NA 1.4 with immersion

    Article Snippet: Once the microsphere was in focus, 300 images were collected at approximately 1 frame per second of this single image plane using an Olympus 60× NA 1.4 oil immersion objective.

    Techniques: Fluorescence

    Axial intensity distribution FWHM of 0.2 μm fluorescent microspheres mounted in agarose with varying refractive index versus NFP. Images collected with 60× oil immersion objective NA 1.4 with immersion oil (refractive index 1.515). Lines

    Journal: Journal of microscopy

    Article Title: The effects of spherical aberration on multiphoton fluorescence excitation microscopy

    doi: 10.1111/j.1365-2818.2010.03449.x

    Figure Lengend Snippet: Axial intensity distribution FWHM of 0.2 μm fluorescent microspheres mounted in agarose with varying refractive index versus NFP. Images collected with 60× oil immersion objective NA 1.4 with immersion oil (refractive index 1.515). Lines

    Article Snippet: Once the microsphere was in focus, 300 images were collected at approximately 1 frame per second of this single image plane using an Olympus 60× NA 1.4 oil immersion objective.

    Techniques:

    Photobleaching rate normalized at the surface of the sample versus NFP. Images were collected with 60× oil immersion objective NA 1.4 with immersion oil (refractive index 1.515) for 5 min of 0.2 μm fluorescent microspheres mounted in agarose

    Journal: Journal of microscopy

    Article Title: The effects of spherical aberration on multiphoton fluorescence excitation microscopy

    doi: 10.1111/j.1365-2818.2010.03449.x

    Figure Lengend Snippet: Photobleaching rate normalized at the surface of the sample versus NFP. Images were collected with 60× oil immersion objective NA 1.4 with immersion oil (refractive index 1.515) for 5 min of 0.2 μm fluorescent microspheres mounted in agarose

    Article Snippet: Once the microsphere was in focus, 300 images were collected at approximately 1 frame per second of this single image plane using an Olympus 60× NA 1.4 oil immersion objective.

    Techniques:

    Signal attenuation and excitation attenuation versus NFP measured in agarose samples with refractive index 1.342 using a 60× oil immersion objective NA 1.4 with immersion oil (refractive index 1.515). Signal attenuation data are the average of

    Journal: Journal of microscopy

    Article Title: The effects of spherical aberration on multiphoton fluorescence excitation microscopy

    doi: 10.1111/j.1365-2818.2010.03449.x

    Figure Lengend Snippet: Signal attenuation and excitation attenuation versus NFP measured in agarose samples with refractive index 1.342 using a 60× oil immersion objective NA 1.4 with immersion oil (refractive index 1.515). Signal attenuation data are the average of

    Article Snippet: Once the microsphere was in focus, 300 images were collected at approximately 1 frame per second of this single image plane using an Olympus 60× NA 1.4 oil immersion objective.

    Techniques:

    Signal attenuation versus NFP of 0.2 μm fluorescent microspheres mounted in agarose with refractive index 1.404 or kidney tissue. Images were collected with a 60× oil immersion objective NA 1.4 with immersion oil (refractive index 1.515).The

    Journal: Journal of microscopy

    Article Title: The effects of spherical aberration on multiphoton fluorescence excitation microscopy

    doi: 10.1111/j.1365-2818.2010.03449.x

    Figure Lengend Snippet: Signal attenuation versus NFP of 0.2 μm fluorescent microspheres mounted in agarose with refractive index 1.404 or kidney tissue. Images were collected with a 60× oil immersion objective NA 1.4 with immersion oil (refractive index 1.515).The

    Article Snippet: Once the microsphere was in focus, 300 images were collected at approximately 1 frame per second of this single image plane using an Olympus 60× NA 1.4 oil immersion objective.

    Techniques:

    Pronephric morphogenesis, patterning, and segmentation in control and morphant embryos. (A–D) Expression of pax2.1 in 1-dpf embryos or cdh17 in 2-dpf embryos as revealed by in situ hybridization. (E, F) Low magnification overview of Na + /K + -ATPase expression in the PTs of 2-dpf control or morphant embryos. (G–P) Expression of wt1a (G–H), nph2s (I, M), slc4a4 (J,N), slc12a (K, O) or clck (L, P) in control or morphant 3-dpf embryos.

    Journal: PLoS ONE

    Article Title: Requirement for a Uroplakin 3a-Like Protein in the Development of Zebrafish Pronephric Tubule Epithelial Cell Function, Morphogenesis, and Polarity

    doi: 10.1371/journal.pone.0041816

    Figure Lengend Snippet: Pronephric morphogenesis, patterning, and segmentation in control and morphant embryos. (A–D) Expression of pax2.1 in 1-dpf embryos or cdh17 in 2-dpf embryos as revealed by in situ hybridization. (E, F) Low magnification overview of Na + /K + -ATPase expression in the PTs of 2-dpf control or morphant embryos. (G–P) Expression of wt1a (G–H), nph2s (I, M), slc4a4 (J,N), slc12a (K, O) or clck (L, P) in control or morphant 3-dpf embryos.

    Article Snippet: The α6F monoclonal antibody supernatant to the Na+ /K+ -ATPase was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA) and used at a 1∶50 dilution.

    Techniques: Expressing, In Situ Hybridization

    Expression of upk3l in the zebrafish pronephros. (A) Dendrogram of the UP3 family (TreeFam accession TF336628 ) Legend: Bt, Bos taurus ; Cf, Canis familiaris ; Dr: Danio rerio ; Gg, Gallus gallus ; Hs, Homo sapiens ; Md, Monodelphis domestica ; Mm, Mus musculus ; Mu, Macaca mulatta ; Pt, Pan troglodytes ; Rn, Rattus norvegicus ; Xt, Xenopus tropicalis . Upk3l (A2CE76) and human (hsUP3a) are highlighted. (B) TMHMM plot and predicted structure of Upk3l: CT, cytosolic tail; EL, extracellular loop; TM, transmembrane domain. (C) Expression of upk3l message in 1-dpf and 2-dpf embryos as detected by RT-PCR; control: reaction in which the RNA template was substituted with water. (D) Western blot analysis of Upk3l expression in uninjected embryos or those injected with upk3l -MO. Acetylated-tubulin (Ac-tub) was used as a loading control. (E) Localization of Upk3l, Na + /K + -ATPase, and nuclei in cross sections of 2-dpf embryos. The Upk3l primary antibody was added to the samples in the upper row of images, but excluded from those in the lower set. The approximate location of the sections is shown in the cartoon above. Legend: G, glomerulus; N, neck; PCT, proximal convoluted tubule; PST, proximal straight tubule; DE, distal early tubule; DL, distal late tubule; CD, collecting duct. (F) Distribution of Upk3l, Na + /K + -ATPase, and nuclei in the PTs of 2-dpf embryos incubated with control MO (upper panels) or upk3l -MO (lower panels). The lumen is marked with an asterisk. (G) Immunolabeling of MDCK cells transiently transfected with cDNA encoding upk3l , human (h)UP3a alone, or hUP3a and hUP1b. Samples were costained with TRITC-phalloidin to label the cortical actin cytoskeleton and/or TO-PRO-3 to label the nuclei. Images are XZ confocal sections.

    Journal: PLoS ONE

    Article Title: Requirement for a Uroplakin 3a-Like Protein in the Development of Zebrafish Pronephric Tubule Epithelial Cell Function, Morphogenesis, and Polarity

    doi: 10.1371/journal.pone.0041816

    Figure Lengend Snippet: Expression of upk3l in the zebrafish pronephros. (A) Dendrogram of the UP3 family (TreeFam accession TF336628 ) Legend: Bt, Bos taurus ; Cf, Canis familiaris ; Dr: Danio rerio ; Gg, Gallus gallus ; Hs, Homo sapiens ; Md, Monodelphis domestica ; Mm, Mus musculus ; Mu, Macaca mulatta ; Pt, Pan troglodytes ; Rn, Rattus norvegicus ; Xt, Xenopus tropicalis . Upk3l (A2CE76) and human (hsUP3a) are highlighted. (B) TMHMM plot and predicted structure of Upk3l: CT, cytosolic tail; EL, extracellular loop; TM, transmembrane domain. (C) Expression of upk3l message in 1-dpf and 2-dpf embryos as detected by RT-PCR; control: reaction in which the RNA template was substituted with water. (D) Western blot analysis of Upk3l expression in uninjected embryos or those injected with upk3l -MO. Acetylated-tubulin (Ac-tub) was used as a loading control. (E) Localization of Upk3l, Na + /K + -ATPase, and nuclei in cross sections of 2-dpf embryos. The Upk3l primary antibody was added to the samples in the upper row of images, but excluded from those in the lower set. The approximate location of the sections is shown in the cartoon above. Legend: G, glomerulus; N, neck; PCT, proximal convoluted tubule; PST, proximal straight tubule; DE, distal early tubule; DL, distal late tubule; CD, collecting duct. (F) Distribution of Upk3l, Na + /K + -ATPase, and nuclei in the PTs of 2-dpf embryos incubated with control MO (upper panels) or upk3l -MO (lower panels). The lumen is marked with an asterisk. (G) Immunolabeling of MDCK cells transiently transfected with cDNA encoding upk3l , human (h)UP3a alone, or hUP3a and hUP1b. Samples were costained with TRITC-phalloidin to label the cortical actin cytoskeleton and/or TO-PRO-3 to label the nuclei. Images are XZ confocal sections.

    Article Snippet: The α6F monoclonal antibody supernatant to the Na+ /K+ -ATPase was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA) and used at a 1∶50 dilution.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Injection, Incubation, Immunolabeling, Transfection

    Disruption of epithelial polarity in upk3l morphants. (A–B) Localization of Prkcz (A) or Pard3 (B), Na + /K + -ATPase, and nuclei in cross-sectioned embryos treated with control (CNT-MO) or upk3l MO ( upk3l -MO). (C) Distribution of Na + /K + -ATPase and nuclei in 2-dpf control or morphant embryos. (D–E) TEM analysis of the proximal segment of PTs from control or morphant embryos. (D) Cross section of tubule show that it is comprised of 5–6 cells surrounding a central lumen. (E) At higher magnification, control lumens were filled with microvilli (mv) and cilia (ci), whereas microvilli were absent in morphants. (F) Immunolocalization of phosphorylated ERM proteins (P-ERM) in frozen sections of embryos. Sections were co-stained for Na + /K + -ATPase and nuclei. (G) Immunostaining of cilia-associated acetylated-tubulin and nuclei in the PT of laterally oriented embryos. p: proximal tubule, d: distal tubule.

    Journal: PLoS ONE

    Article Title: Requirement for a Uroplakin 3a-Like Protein in the Development of Zebrafish Pronephric Tubule Epithelial Cell Function, Morphogenesis, and Polarity

    doi: 10.1371/journal.pone.0041816

    Figure Lengend Snippet: Disruption of epithelial polarity in upk3l morphants. (A–B) Localization of Prkcz (A) or Pard3 (B), Na + /K + -ATPase, and nuclei in cross-sectioned embryos treated with control (CNT-MO) or upk3l MO ( upk3l -MO). (C) Distribution of Na + /K + -ATPase and nuclei in 2-dpf control or morphant embryos. (D–E) TEM analysis of the proximal segment of PTs from control or morphant embryos. (D) Cross section of tubule show that it is comprised of 5–6 cells surrounding a central lumen. (E) At higher magnification, control lumens were filled with microvilli (mv) and cilia (ci), whereas microvilli were absent in morphants. (F) Immunolocalization of phosphorylated ERM proteins (P-ERM) in frozen sections of embryos. Sections were co-stained for Na + /K + -ATPase and nuclei. (G) Immunostaining of cilia-associated acetylated-tubulin and nuclei in the PT of laterally oriented embryos. p: proximal tubule, d: distal tubule.

    Article Snippet: The α6F monoclonal antibody supernatant to the Na+ /K+ -ATPase was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA) and used at a 1∶50 dilution.

    Techniques: Transmission Electron Microscopy, Staining, Immunostaining

    TMEM30a-GFP localizes in the plasma membrane and internal organelles (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask ™ Orange Plasma Membrane to mark the plasma membrane ( top ) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum ( row 2 ), or Golgi ( row 3 ). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria ( bottom ). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

    doi: 10.4049/jimmunol.1002710

    Figure Lengend Snippet: TMEM30a-GFP localizes in the plasma membrane and internal organelles (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask ™ Orange Plasma Membrane to mark the plasma membrane ( top ) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum ( row 2 ), or Golgi ( row 3 ). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria ( bottom ). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

    Article Snippet: Anti-Na/K ATPase was from Abcam (Cambridge, MA).

    Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Cytometry, Incubation

    Schematic representation summarizing the transforming growth factors-beta (TGF-β) receptor complexes and signaling pathway in endothelial cells. The green arrowhead lines represent positive crosstalk interactions and steps. Red flat-ended lines indicate inhibition. In the nucleus, the green arrowhead and red flat-ended lines on the DNA represent activation and inhibition of gene expression, respectively. ( a ) LAP-TGF-β latent complex. ( b ) Release of TGF-β from the complex with LAP. ( c ) TGF-β binding to the heterotetrameric receptor complex. ( d ) TβRII phosphorylation of type I receptor ALK5. ( e ) SMAD2/3 phosphorylation by ALK5. ( f ) SMAD1/5/8 phosphorylation by ALK1. ( g ) Dimerization of R-SMADs with SMAD4. ( h ) Translocation of the R-SMAD/SMAD4 complex into the nucleus and binding to regulatory sequences. See text for details.

    Journal: International Journal of Molecular Sciences

    Article Title: The Controversial Role of TGF-β in Neovascular Age-Related Macular Degeneration Pathogenesis

    doi: 10.3390/ijms19113363

    Figure Lengend Snippet: Schematic representation summarizing the transforming growth factors-beta (TGF-β) receptor complexes and signaling pathway in endothelial cells. The green arrowhead lines represent positive crosstalk interactions and steps. Red flat-ended lines indicate inhibition. In the nucleus, the green arrowhead and red flat-ended lines on the DNA represent activation and inhibition of gene expression, respectively. ( a ) LAP-TGF-β latent complex. ( b ) Release of TGF-β from the complex with LAP. ( c ) TGF-β binding to the heterotetrameric receptor complex. ( d ) TβRII phosphorylation of type I receptor ALK5. ( e ) SMAD2/3 phosphorylation by ALK5. ( f ) SMAD1/5/8 phosphorylation by ALK1. ( g ) Dimerization of R-SMADs with SMAD4. ( h ) Translocation of the R-SMAD/SMAD4 complex into the nucleus and binding to regulatory sequences. See text for details.

    Article Snippet: However, in the latter studies it is not clear which TGF-β isoform was specifically detected, because, in the immunolocalization experiments, the authors used a “panspecific” polyclonal antibody (AB-100-NA, from R & D Systems) raised in rabbit against a mixture containing recombinant human TGF-β1 and porcine TGF-β2, without any specification of isoform affinity or evidence for human TGF-β2 recognition.

    Techniques: Inhibition, Activation Assay, Expressing, Binding Assay, Translocation Assay

    IL-6 and BMP-6 induces AR activation in LNCaP cells (a) HTS33B and HPS33Q human prostate stromal cells were cultured in RPMI1640 supplemented with 0.2% charcoal-stripped FBS and treated with TGF-β1 (50 pM) for 3 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of IL-6 and BMP-6. Data is from three independent experiments. (b) LNCaP cells were co-cultured with HTS33B cells in RPMI1640 supplemented with 0.2% charcoal-stripped FBS, and treated with TGF-β1 (50 pM), IL-6 (25 ng/ml), and/or BMP-6 (50 ng/ml) for 6 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of PSA, KLK4, TMPRSS2 expresssion in these co-cultures. Data is from three independent experiments. (c) LNCaP cells were similarly co-cultured with HPS33Q cells and treated with TGF-β1 (50 pM), IL-6 (25 ng/ml), and/or BMP-6 (50 ng/ml) for 6 days, and analyzed for the expression of PSA, KLK4, TMPRSS2 (qPCR). Data is from three independent experiments. All gene expression data were normalized to GAPDH expression. hPS (human prostate stromal cells) stands for either HTS33B (b) or HPS33Q cells (c) . *p

    Journal: Oncotarget

    Article Title: Stromal TGF-β signaling induces AR activation in prostate cancer

    doi:

    Figure Lengend Snippet: IL-6 and BMP-6 induces AR activation in LNCaP cells (a) HTS33B and HPS33Q human prostate stromal cells were cultured in RPMI1640 supplemented with 0.2% charcoal-stripped FBS and treated with TGF-β1 (50 pM) for 3 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of IL-6 and BMP-6. Data is from three independent experiments. (b) LNCaP cells were co-cultured with HTS33B cells in RPMI1640 supplemented with 0.2% charcoal-stripped FBS, and treated with TGF-β1 (50 pM), IL-6 (25 ng/ml), and/or BMP-6 (50 ng/ml) for 6 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of PSA, KLK4, TMPRSS2 expresssion in these co-cultures. Data is from three independent experiments. (c) LNCaP cells were similarly co-cultured with HPS33Q cells and treated with TGF-β1 (50 pM), IL-6 (25 ng/ml), and/or BMP-6 (50 ng/ml) for 6 days, and analyzed for the expression of PSA, KLK4, TMPRSS2 (qPCR). Data is from three independent experiments. All gene expression data were normalized to GAPDH expression. hPS (human prostate stromal cells) stands for either HTS33B (b) or HPS33Q cells (c) . *p

    Article Snippet: Reagents The Porcine TGF-β1(204-B-002), recombinant human BMP-6 (507-BP), recombinant human IL-6 (206-IL-010), antibodies against human BMP-6 (AF-507), human IL-6 (AF-206-NA), and normal goat IgG (AB-108-C) were purchased from R & D System (Minneapolis, MN, USA).

    Techniques: Activation Assay, Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    IL-6 and BMP-6 neutralizing antibody treatment attenuates stromal TGF-β induced AR activation in the co-cultured LNCaP cells (a) LNCaP cells were cultured alone or co-cultured with HTS33B cells in RPMI1640 supplemented with 0.2% charcoal-stripped FBS, and treated with TGF-β1 (50 pM) along with neutralizing antibody against IL-6 and/or BMP-6 (both at 2 μg/ml) for 6 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of PSA, KLK4, TMPRSS2 expresssion in these co-cultures. Data is from two independent experiments. (b) LNCaP cells were similarly cultured alone or co-cultured with HPS33Q cells and treated with TGF-β1 (50 pM) along with neutralizing antibody against IL-6 and/or BMP-6 (both at 2 μg/ml) for 6 days, and analyzed for the expression of PSA, KLK4, TMPRSS2 (qPCR). Data is from two independent experiments. All gene expression data were normalized to GAPDH expression. *p

    Journal: Oncotarget

    Article Title: Stromal TGF-β signaling induces AR activation in prostate cancer

    doi:

    Figure Lengend Snippet: IL-6 and BMP-6 neutralizing antibody treatment attenuates stromal TGF-β induced AR activation in the co-cultured LNCaP cells (a) LNCaP cells were cultured alone or co-cultured with HTS33B cells in RPMI1640 supplemented with 0.2% charcoal-stripped FBS, and treated with TGF-β1 (50 pM) along with neutralizing antibody against IL-6 and/or BMP-6 (both at 2 μg/ml) for 6 days. Total RNA was extracted and reverse transcribed. qPCR were used to analyze the expression of PSA, KLK4, TMPRSS2 expresssion in these co-cultures. Data is from two independent experiments. (b) LNCaP cells were similarly cultured alone or co-cultured with HPS33Q cells and treated with TGF-β1 (50 pM) along with neutralizing antibody against IL-6 and/or BMP-6 (both at 2 μg/ml) for 6 days, and analyzed for the expression of PSA, KLK4, TMPRSS2 (qPCR). Data is from two independent experiments. All gene expression data were normalized to GAPDH expression. *p

    Article Snippet: Reagents The Porcine TGF-β1(204-B-002), recombinant human BMP-6 (507-BP), recombinant human IL-6 (206-IL-010), antibodies against human BMP-6 (AF-507), human IL-6 (AF-206-NA), and normal goat IgG (AB-108-C) were purchased from R & D System (Minneapolis, MN, USA).

    Techniques: Activation Assay, Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    Activated neutrophils are more sensitive to NK cell cytotoxicity compared to resting neutrophils. (A) Percentage of dead neutrophils (PMN) after a 3 h co-culture of neutrophils [pre-stimulated in vitro with CL097 and GM-CSF, or kept on ice (ctrl)] and bulk-NK cells at an E:T ratio of 10:1, with addition of an HLA antibody as indicated ( n = 7; one-way ANOVA followed by Dunnett's multiple comparisons test). (B) Graph shows percentage of dead neutrophils after a 4 h cytotoxicity assay performed using autologous NK effector cells toward transmigrated (ePMN) or blood neutrophils (bPMN; E:T ratio 10:1; n = 5; one-way ANOVA followed by Dunnett's multiple comparisons test). Error bars represent SEM. (C) Representative FACS plots showing percentage of dead neutrophils (transmigrated or resting) measured using AnnexinV and To-Pro-3, after a 4 h co-culture with autologous NK cells.

    Journal: Frontiers in Immunology

    Article Title: Downregulation of HLA Class I Renders Inflammatory Neutrophils More Susceptible to NK Cell-Induced Apoptosis

    doi: 10.3389/fimmu.2019.02444

    Figure Lengend Snippet: Activated neutrophils are more sensitive to NK cell cytotoxicity compared to resting neutrophils. (A) Percentage of dead neutrophils (PMN) after a 3 h co-culture of neutrophils [pre-stimulated in vitro with CL097 and GM-CSF, or kept on ice (ctrl)] and bulk-NK cells at an E:T ratio of 10:1, with addition of an HLA antibody as indicated ( n = 7; one-way ANOVA followed by Dunnett's multiple comparisons test). (B) Graph shows percentage of dead neutrophils after a 4 h cytotoxicity assay performed using autologous NK effector cells toward transmigrated (ePMN) or blood neutrophils (bPMN; E:T ratio 10:1; n = 5; one-way ANOVA followed by Dunnett's multiple comparisons test). Error bars represent SEM. (C) Representative FACS plots showing percentage of dead neutrophils (transmigrated or resting) measured using AnnexinV and To-Pro-3, after a 4 h co-culture with autologous NK cells.

    Article Snippet: Goat serum and α1-antitrypsin were obtained from Sigma-Aldrich (MO, USA), CL097 from InvivoGen (San Diego, CA), GM-CSF from R & D Systems (MN, USA), anti-PR3 from Abcam (UK), and Batimastat and cathepsin G inhibitor from Calbiochem (CA, USA).

    Techniques: Co-Culture Assay, In Vitro, Cytotoxicity Assay, FACS

    NKR ligand expression on in vitro -activated neutrophils. (A) Surface expression of HLA-ABC, CD11b, CD66 or CD62L as indicated, on resting neutrophils kept on ice (ctrl) or in vitro -activated neutrophils stimulated for 20 min at 37°C alone or in presence of the TLR-agonist CL097 and GM-CSF (one-way ANOVA followed by Dunnett's multiple comparisons test). (B) Surface expression of indicated ligands to NKRs on resting neutrophils (ctrl) or in vitro -activated neutrophils stimulated for 20 min at 37°C with CL097/GM-CSF (paired t -test). (C) Expression of NKR ligands and neutrophil degranulation markers on resting neutrophils (ctrl) or in vitro -activated neutrophils stimulated for 1, 2, or 4 h at 37°C with CL097/GM-CSF (one-way ANOVA followed by Dunnett's multiple comparisons test). Error bars represent SEM.

    Journal: Frontiers in Immunology

    Article Title: Downregulation of HLA Class I Renders Inflammatory Neutrophils More Susceptible to NK Cell-Induced Apoptosis

    doi: 10.3389/fimmu.2019.02444

    Figure Lengend Snippet: NKR ligand expression on in vitro -activated neutrophils. (A) Surface expression of HLA-ABC, CD11b, CD66 or CD62L as indicated, on resting neutrophils kept on ice (ctrl) or in vitro -activated neutrophils stimulated for 20 min at 37°C alone or in presence of the TLR-agonist CL097 and GM-CSF (one-way ANOVA followed by Dunnett's multiple comparisons test). (B) Surface expression of indicated ligands to NKRs on resting neutrophils (ctrl) or in vitro -activated neutrophils stimulated for 20 min at 37°C with CL097/GM-CSF (paired t -test). (C) Expression of NKR ligands and neutrophil degranulation markers on resting neutrophils (ctrl) or in vitro -activated neutrophils stimulated for 1, 2, or 4 h at 37°C with CL097/GM-CSF (one-way ANOVA followed by Dunnett's multiple comparisons test). Error bars represent SEM.

    Article Snippet: Goat serum and α1-antitrypsin were obtained from Sigma-Aldrich (MO, USA), CL097 from InvivoGen (San Diego, CA), GM-CSF from R & D Systems (MN, USA), anti-PR3 from Abcam (UK), and Batimastat and cathepsin G inhibitor from Calbiochem (CA, USA).

    Techniques: Expressing, In Vitro

    Downmodulation of HLA-C and HLA-E on activated neutrophils. (A–D) Neutrophil staining of indicated HLA class I cell surface structures (HLA-C or HLA-E) binding to inhibitory NKRs. Resting neutrophils were kept on ice (ctrl) or stimulated for 20 min with the TLR-agonist CL097 and GM-CSF at 37°C ( A ; paired t -test) or for 1, 2, or 4 h under the same conditions ( D ; one-way ANOVA followed by Dunnett's multiple comparisons test). (B) Representative staining of HLA-C and HLA-E of resting neutrophils (gray) or neutrophils that had been stimulated with CL097/GM-CSF for 20 min (orange). (C) Representative staining from one experiment showing expression of HLA-C on resting neutrophils (gray) or neutrophils that had been stimulated with CL097/GM-CSF for 1 h (red). (E) Neutrophil staining of indicated HLA class I molecules on transmigrated neutrophils from skin chamber exudates (ePMN) or resting autologous neutrophils (bPMN; paired t -test). (F) Representative staining from one experiment showing the expression of HLA-C on resting neutrophils (gray) or transmigrated neutrophils (green). Error bars represent SEM.

    Journal: Frontiers in Immunology

    Article Title: Downregulation of HLA Class I Renders Inflammatory Neutrophils More Susceptible to NK Cell-Induced Apoptosis

    doi: 10.3389/fimmu.2019.02444

    Figure Lengend Snippet: Downmodulation of HLA-C and HLA-E on activated neutrophils. (A–D) Neutrophil staining of indicated HLA class I cell surface structures (HLA-C or HLA-E) binding to inhibitory NKRs. Resting neutrophils were kept on ice (ctrl) or stimulated for 20 min with the TLR-agonist CL097 and GM-CSF at 37°C ( A ; paired t -test) or for 1, 2, or 4 h under the same conditions ( D ; one-way ANOVA followed by Dunnett's multiple comparisons test). (B) Representative staining of HLA-C and HLA-E of resting neutrophils (gray) or neutrophils that had been stimulated with CL097/GM-CSF for 20 min (orange). (C) Representative staining from one experiment showing expression of HLA-C on resting neutrophils (gray) or neutrophils that had been stimulated with CL097/GM-CSF for 1 h (red). (E) Neutrophil staining of indicated HLA class I molecules on transmigrated neutrophils from skin chamber exudates (ePMN) or resting autologous neutrophils (bPMN; paired t -test). (F) Representative staining from one experiment showing the expression of HLA-C on resting neutrophils (gray) or transmigrated neutrophils (green). Error bars represent SEM.

    Article Snippet: Goat serum and α1-antitrypsin were obtained from Sigma-Aldrich (MO, USA), CL097 from InvivoGen (San Diego, CA), GM-CSF from R & D Systems (MN, USA), anti-PR3 from Abcam (UK), and Batimastat and cathepsin G inhibitor from Calbiochem (CA, USA).

    Techniques: Staining, Binding Assay, Expressing

    Decrease of surface HLA class I molecules is not explained by internalization. Graph shows resting neutrophils stained with FITC-conjugated HLA-ABC antibody prior to addition of buffer (ctrl) or CL097/GM-CSF for 20 min at 37°C, after which FITC-quenching antibody was added.

    Journal: Frontiers in Immunology

    Article Title: Downregulation of HLA Class I Renders Inflammatory Neutrophils More Susceptible to NK Cell-Induced Apoptosis

    doi: 10.3389/fimmu.2019.02444

    Figure Lengend Snippet: Decrease of surface HLA class I molecules is not explained by internalization. Graph shows resting neutrophils stained with FITC-conjugated HLA-ABC antibody prior to addition of buffer (ctrl) or CL097/GM-CSF for 20 min at 37°C, after which FITC-quenching antibody was added.

    Article Snippet: Goat serum and α1-antitrypsin were obtained from Sigma-Aldrich (MO, USA), CL097 from InvivoGen (San Diego, CA), GM-CSF from R & D Systems (MN, USA), anti-PR3 from Abcam (UK), and Batimastat and cathepsin G inhibitor from Calbiochem (CA, USA).

    Techniques: Staining