β gal Search Results


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  • 98
    Thermo Fisher β galactosidase β gal
    The effect of Box A, Box C and Box B mutations on origin-dependent DNA replication and flanking gene expression in the context of VZV-MSP superinfection. A) A typical Southern blot analysis of the effects on DNA replication of CGC motif mutation in Box A, C and B to AAA. The upper band (R) indicates the position of DpnI resistant DNA resulting from replication in MeWo cells. The lower band (U) indicates the position of unreplicated input plasmid. B) A histogram summarizing the data from three independent DpnI replication assays analyzed at 48 hrs post VZV-MSP superinfection. C) Results of triplicate assays comparing the effect of the presence of Box A, Box C and Box B mutations on the expression levels of the Renilla luciferase reporter gene present at the position of ORF62 and D) the firefly luciferase reporter gene present at the position of ORF63. The promoter activities in the presence of VZV-MSP superinfection are represented as <t>RLU/β-Gal</t> units. The black and grey bars represent experiments done in the absence and presence of 400 μg/ml of PAA respectively. Statistical significance was determined by a oneway ANOVA analysis of variance followed by Tukey's post hoc test.
    β Galactosidase β Gal, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β galactosidase β gal
    (A) A minimal domain of NS3 required for interaction was defined using the yeast two-hybrid assay. N- and C-terminal truncations in NS3 were made, and the fragments were cloned into pAS2–1. These were transformed into PJ69–2A, and transformants were mated with Y187 carrying pACT-NS3. The resulting diploids were patched onto −Trp −Leu plates, replica plated onto −His and −Ade plates, and also lifted onto a nylon membrane for the <t>β-Gal</t> assay. A fragment encompassing aa 182 to 335 near the N terminus of the helicase domain, which includes conserved residues important for NTP-binding, NTPase, and helicase activities, was defined to be the minimal region required for interaction with NS3. Positive interactions were indicated by growth on −His and −Ade plates and by the presence of β-Gal activity. (B) IP between Flag-NS3 and myc-tagged NS3 proteins. COS cells were transfected with Flag-NS3 and myc-NS3 or with myc-NS3 alone. Total protein (100 μg) from transfected cells was used for IP using an anti-Flag agarose gel. Lane 1, myc-NS3 was detected in the complex precipitated with an anti-Flag gel, demonstrating interaction between myc-NS3 and Flag-NS3. Lane 2, myc-NS3 was not precipitated by the anti-Flag gel in the absence of Flag-NS3, showing the specificity of the anti-Flag gel used for IP. Lanes 3 and 4, 1/10 of the input IP proteins was probed with anti-Flag antibody to show that Flag-NS3 was specifically precipitated by the anti-Flag gel. Lanes 5 through 7, Western blot of total cell lysate (20 μg loaded) to detect the expression of each of the tagged NS3 proteins in transfected cells. IgG, immunoglobulin G.
    β Galactosidase β Gal, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Biogenesis Inc β galactosidase β gal
    A large portion of neurons infected in caudal VTA project to the NAc and are dopaminergic. The number of <t>β-gal-positive</t> neurons did not significantly differ between rostral and caudal VTA. However, more neurons were retrogradely labeled in caudal VTA than in rostral VTA after injection of HS in to the NAc. More neurons in caudal VTA than in rostral VTA also were colabeled with β-gal and HS. Most of the HS- and β-gal-positive neurons in rostral and caudal VTA were also TH positive. B-G , A representative section from caudal VTA, triple labeled for HS ( B ), β-gal ( C ), and TH ( D ). E , Merged image from B and C showing neurons colabeled with HS and β-gal. F , Merged image from B and D showing neurons colabeled with HS and TH. G , Merged image from B-D showing triple-labeled neurons (arrows). Data are expressed as cell numbers per section ± SEM ( n = 3). * p
    β Galactosidase β Gal, supplied by Biogenesis Inc, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa β galactosidase β gal
    Increased Ca 2 + influx into and CK efflux from BIO14.6 myotubes and their correction by δ-SG gene transfer. (a) 45 Ca 2+ uptake into normal or BIO14.6 myotubes measured under resting conditions with or without SK F96365 (SK F), ruthenium red (RR), or Nifedipine. The Gd 3+ -inhibitable fractions are shown. (b) External Ca 2+ -induced changes in fluo-4 fluorescence in normal or BIO14.6 myotubes. The bar graph shows the maximal increments of fluorescence in myotubes pretreated with or without 50 μM SK F96365. ΔF/F 0 is the ratio between the fluorescence increment and the fluorescence before Ca 2+ addition. Numbers in parentheses correspond to the number of cells studied. (c) CK efflux from BIO14.6 myotubes subjected to cyclic stretch under indicated conditions. (d) Immunoblot assay and immunohistochemistry (IH) of BIO14.6 myotubes infected with <t>Ad.β-gal</t> or Ad.δ-SG. (e and f) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch–induced CK efflux in BIO14.6 myotubes insfected with Ad.β-gal or Ad.δ-SG. TG, thapsigargin (1 μM). Other conditions were similar to b and c. In these panels, error bars show means ± SD and asterisks show P
    β Galactosidase β Gal, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DiscoverX β galactosidase β gal substrate
    Increased Ca 2 + influx into and CK efflux from BIO14.6 myotubes and their correction by δ-SG gene transfer. (a) 45 Ca 2+ uptake into normal or BIO14.6 myotubes measured under resting conditions with or without SK F96365 (SK F), ruthenium red (RR), or Nifedipine. The Gd 3+ -inhibitable fractions are shown. (b) External Ca 2+ -induced changes in fluo-4 fluorescence in normal or BIO14.6 myotubes. The bar graph shows the maximal increments of fluorescence in myotubes pretreated with or without 50 μM SK F96365. ΔF/F 0 is the ratio between the fluorescence increment and the fluorescence before Ca 2+ addition. Numbers in parentheses correspond to the number of cells studied. (c) CK efflux from BIO14.6 myotubes subjected to cyclic stretch under indicated conditions. (d) Immunoblot assay and immunohistochemistry (IH) of BIO14.6 myotubes infected with <t>Ad.β-gal</t> or Ad.δ-SG. (e and f) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch–induced CK efflux in BIO14.6 myotubes insfected with Ad.β-gal or Ad.δ-SG. TG, thapsigargin (1 μM). Other conditions were similar to b and c. In these panels, error bars show means ± SD and asterisks show P
    β Galactosidase β Gal Substrate, supplied by DiscoverX, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher β galactosidase β gal plasmid
    miR-218-5p directly downregulates EGFR in NSCLC cells A. Schematic representation of the putative miR-218-5p binding sequence on the 3′-UTR of the EGFR mRNA. B. The luciferase reporter plasmid encoding the full-length 3′-UTR of EGFR was co-transfected into the A549 and H1975 cells along with miR-218-5p mimic or scrambled mimic. The luciferase activities were normalized to the <t>β-galactosidase</t> levels of the control (**P
    β Galactosidase β Gal Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pcmv β galactosidase β gal
    miR-218-5p directly downregulates EGFR in NSCLC cells A. Schematic representation of the putative miR-218-5p binding sequence on the 3′-UTR of the EGFR mRNA. B. The luciferase reporter plasmid encoding the full-length 3′-UTR of EGFR was co-transfected into the A549 and H1975 cells along with miR-218-5p mimic or scrambled mimic. The luciferase activities were normalized to the <t>β-galactosidase</t> levels of the control (**P
    Pcmv β Galactosidase β Gal, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Aldevron β galactosidase β gal gene
    ( A ) Extents of expression in COS-7 cell cultures of the <t>β-gal</t> gene mediated in the presence of serum by PEI2 (bars a–c) and PEI2–GNPII (bars d–f) at different N/P ratios. Bars a and d, N/P = 90; bars b and e, N/P = 120; bars c and f, N/P = 150. ( B ) Extents of expression in COS-7 cell cultures of the β-gal gene mediated in the presence of serum by PEI25 (bar a), dodecyl–PEI2 (bars b and c), PEI2–GNPII (bar d), and PEI2–GNPII + dodecyl–PEI2 (bars e and f). Bar a, N/P = 10; bar b, N/P = 20; bar c, N/P = 40; bar d, N/P = 150; bar e, N/P = 150 + 20; bar f, N/P = 150 + 40. Cells transfected with the plasmid in the absence of polycations showed no appreciable β-gal activity.
    β Galactosidase β Gal Gene, supplied by Aldevron, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher β galactosidase β gal reagent
    ( A ) Extents of expression in COS-7 cell cultures of the <t>β-gal</t> gene mediated in the presence of serum by PEI2 (bars a–c) and PEI2–GNPII (bars d–f) at different N/P ratios. Bars a and d, N/P = 90; bars b and e, N/P = 120; bars c and f, N/P = 150. ( B ) Extents of expression in COS-7 cell cultures of the β-gal gene mediated in the presence of serum by PEI25 (bar a), dodecyl–PEI2 (bars b and c), PEI2–GNPII (bar d), and PEI2–GNPII + dodecyl–PEI2 (bars e and f). Bar a, N/P = 10; bar b, N/P = 20; bar c, N/P = 40; bar d, N/P = 150; bar e, N/P = 150 + 20; bar f, N/P = 150 + 40. Cells transfected with the plasmid in the absence of polycations showed no appreciable β-gal activity.
    β Galactosidase β Gal Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher pcmv β galactosidase β gal
    NS1 containing cell culture supernatants inhibit the TLR3 response in HeLa cells. (A) HeLa cells were transfected with an IFNβ-Luc reporter construct and a <t>β-gal</t> expressing plasmid and subsequently co-cultured with either regular HeLa cells or HeLa cells harboring a WNV replicon (HeLa rep). Luc activity was determined 4h after the addition of pIC to the culture media and normalized to β-gal activity. Asterisks represent statistically significant differences, determined by student’s t-test, comparing the pIC response of HeLa:HeLa rep samples to that of HeLa:HeLa samples. (B) HeLa cells transfected with IFNβ reporter and β-gal as above were incubated for 16h with cell culture supernatants from either regular HeLa cells or HeLa rep cells then stimulated with pIC before Luc activity was determined. Asterisks represent statistically significant differences, determined by student’s t-test comparing the pIC response of HeLa rep cells to that of HeLa cells. (C) HeLa cells were incubated with cell culture supernatants from either 293T-Ctrl or 293T-NS1 cells for 16h. NS1 association with the cells was detected by immunofluorescence of permeabilized cells using WNV MHIAF. (D) HeLa cells were incubated with control or sNS1-containing supernatants for 16h and extensively washed before lysis. Whole cell lysates were analyzed by immunoblot with WNV MHIAF followed by HRP-labeled mouse antibody. (E) NS1 association with naïve cells by flow cytometry. HeLa cells were incubated with control or sNS1-containing supernatants for 6h and 24h either left unpermeabilized to detect NS1 on the cell surface or permeabilized prior to staining to detect internalized NS1. NS1 was detected with an NS1-specific antibody. (F) Secreted NS1-containing cell culture supernatants inhibit the TLR3 response in HeLa cells. HeLa cells transfected with IFNβ reporter construct and β-gal were incubated with supernatants from either 293T-Ctrl or 293T-NS1 cells for 16h and stimulated as above. Asterisks represent statistically significant differences, determined by student’s t-test of sNS1-containing supernatant treated samples compared to control treated samples
    Pcmv β Galactosidase β Gal, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega β galactosidase β gal reagents
    NS1 containing cell culture supernatants inhibit the TLR3 response in HeLa cells. (A) HeLa cells were transfected with an IFNβ-Luc reporter construct and a <t>β-gal</t> expressing plasmid and subsequently co-cultured with either regular HeLa cells or HeLa cells harboring a WNV replicon (HeLa rep). Luc activity was determined 4h after the addition of pIC to the culture media and normalized to β-gal activity. Asterisks represent statistically significant differences, determined by student’s t-test, comparing the pIC response of HeLa:HeLa rep samples to that of HeLa:HeLa samples. (B) HeLa cells transfected with IFNβ reporter and β-gal as above were incubated for 16h with cell culture supernatants from either regular HeLa cells or HeLa rep cells then stimulated with pIC before Luc activity was determined. Asterisks represent statistically significant differences, determined by student’s t-test comparing the pIC response of HeLa rep cells to that of HeLa cells. (C) HeLa cells were incubated with cell culture supernatants from either 293T-Ctrl or 293T-NS1 cells for 16h. NS1 association with the cells was detected by immunofluorescence of permeabilized cells using WNV MHIAF. (D) HeLa cells were incubated with control or sNS1-containing supernatants for 16h and extensively washed before lysis. Whole cell lysates were analyzed by immunoblot with WNV MHIAF followed by HRP-labeled mouse antibody. (E) NS1 association with naïve cells by flow cytometry. HeLa cells were incubated with control or sNS1-containing supernatants for 6h and 24h either left unpermeabilized to detect NS1 on the cell surface or permeabilized prior to staining to detect internalized NS1. NS1 was detected with an NS1-specific antibody. (F) Secreted NS1-containing cell culture supernatants inhibit the TLR3 response in HeLa cells. HeLa cells transfected with IFNβ reporter construct and β-gal were incubated with supernatants from either 293T-Ctrl or 293T-NS1 cells for 16h and stimulated as above. Asterisks represent statistically significant differences, determined by student’s t-test of sNS1-containing supernatant treated samples compared to control treated samples
    β Galactosidase β Gal Reagents, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher invitrogen β galactosidase β gal
    HSV-1 entry into nTERT cells requires the gD receptor nectin-1. (A and B) Nectin-1 (A) or HVEM (B) mRNA was quantitated in HeLa and nTERT cells by qRT-PCR using the ΔΔ C T method. The housekeeping gene RPLP0 was used to normalize expression of both genes. (C and D) nTERT or HeLa cells were reverse transfected with 30 nM nectin-1, HVEM, or control siRNAs. Seventy-two hours later, cell RNA was extracted from half of the siRNA-transfected cells, and nectin-1 and HVEM mRNA levels were quantitated by qRT-PCR using the ΔΔ C T method. (C) The housekeeping gene RPLP0 was used to normalize expression of both genes. The remaining cells were transferred in triplicate to 96-well plates and incubated for a further 16 h. These cells were infected on ice with 110lacZ virus at a multiplicity of infection of 2 for 1 h prior to replacing the inoculum with fresh media at 37°C and incubating for a further 4 h. Cells were lysed and relative <t>β-galactosidase</t> activity measured. (D) A representative experiment is shown as the means ± standard errors of the data relative to the negative siRNA control.
    Invitrogen β Galactosidase β Gal, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Agilent technologies β galactosidase β gal
    HSV-1 entry into nTERT cells requires the gD receptor nectin-1. (A and B) Nectin-1 (A) or HVEM (B) mRNA was quantitated in HeLa and nTERT cells by qRT-PCR using the ΔΔ C T method. The housekeeping gene RPLP0 was used to normalize expression of both genes. (C and D) nTERT or HeLa cells were reverse transfected with 30 nM nectin-1, HVEM, or control siRNAs. Seventy-two hours later, cell RNA was extracted from half of the siRNA-transfected cells, and nectin-1 and HVEM mRNA levels were quantitated by qRT-PCR using the ΔΔ C T method. (C) The housekeeping gene RPLP0 was used to normalize expression of both genes. The remaining cells were transferred in triplicate to 96-well plates and incubated for a further 16 h. These cells were infected on ice with 110lacZ virus at a multiplicity of infection of 2 for 1 h prior to replacing the inoculum with fresh media at 37°C and incubating for a further 4 h. Cells were lysed and relative <t>β-galactosidase</t> activity measured. (D) A representative experiment is shown as the means ± standard errors of the data relative to the negative siRNA control.
    β Galactosidase β Gal, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare β galactosidase β gal gene
    Activation of HIV-1 gene expression by Tat. HeLa cells were cotransfected with the reporter constructs HIV-1 LTR-CAT and pCH110 <t>(β-Gal)</t> together with plasmids (pDex) that expressed either the β-globin gene (bar 1), the wild type tat gene (bar 2), or the mutated tat genes corresponding to [E2G, D5G, E9G] (bar 3), P3L (bar 4), P[6, 10]L (bar 5), P[10, 14]L (bar 6), C27S (bar 7), K41A (bar 8), and K/R[50-57]G (bar 9). The cells were harvested at 48 h posttransfection, and equal amounts of protein were normalized to β-Gal activity and assayed for CAT protein by ELISA. The transfections were performed three times with the standard deviations indicated.
    β Galactosidase β Gal Gene, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega psv β galactosidase β gal
    Transactivation of PPARγ 1 /RXRγ heterodimers via E- and H-FABP-PPRE without and with ligand ( A ) HepG2 cells were transiently co-transfected with pCAT3 promoter plasmid containing the PPRE, <t>β-Gal,</t> without and with plasmids for PPARγ 1 and RXRγ. After 42 h, cells were harvested, and β-Gal and CAT concentrations were determined by ELISAs. For each PPRE, experiments were normalized to those without ectopic PPARγ 1 /RXRγ, set to unity. Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6). ( B ) HepG2 cells were transiently co-transfected with pCAT3 promoter plasmid containing the PPRE and plasmids for β-Gal, PPARγ 1 and RXRγ. DMSO and DMSO plus bezafibrate respectively were added to the medium 4 h after transfection (final concentrations 1% DMSO and 100 μM bezafibrate) and cells were incubated for a further 38 h. After harvest, β-Gal and CAT concentrations were determined by ELISAs. For each PPRE, experiments with ligand were normalized to those with DMSO alone, set to unity. Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6).
    Psv β Galactosidase β Gal, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega β galactosidase β gal activity
    Transactivation of PPARγ 1 /RXRγ heterodimers via E- and H-FABP-PPRE without and with ligand ( A ) HepG2 cells were transiently co-transfected with pCAT3 promoter plasmid containing the PPRE, <t>β-Gal,</t> without and with plasmids for PPARγ 1 and RXRγ. After 42 h, cells were harvested, and β-Gal and CAT concentrations were determined by ELISAs. For each PPRE, experiments were normalized to those without ectopic PPARγ 1 /RXRγ, set to unity. Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6). ( B ) HepG2 cells were transiently co-transfected with pCAT3 promoter plasmid containing the PPRE and plasmids for β-Gal, PPARγ 1 and RXRγ. DMSO and DMSO plus bezafibrate respectively were added to the medium 4 h after transfection (final concentrations 1% DMSO and 100 μM bezafibrate) and cells were incubated for a further 38 h. After harvest, β-Gal and CAT concentrations were determined by ELISAs. For each PPRE, experiments with ligand were normalized to those with DMSO alone, set to unity. Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6).
    β Galactosidase β Gal Activity, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cortex Biochem Inc β galactosidase β gal
    Dlx2 -expressing cells and their progeny, defined by Dlx2/tauLacZ <t>(β-gal)</t> expression, intermix with Zebrin II+ VZ cells to form the dorsolateral SVZ. A–C , β-gal (TRITC)- and Zebrin II (FITC)-immunoreactive cells begin to mix perinatally. D–I , Postnatally, Zebrin II+ cells are displaced to form the outer limits of the SVZ, whereas β-gal+ cells constitute the Zebrin II− central region at P6 ( D–F ) and P10 ( G–I ). Note that in A and D , β-gal+ cells also stream into the overlying white matter. Scale bars: B , 100 μm; E , 50 μm; H , 20 μm.
    β Galactosidase β Gal, supplied by Cortex Biochem Inc, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Virogen β galactosidase β gal control protein
    HCV core-induced TNF-α and IL-6 mediate the enhancement of HIV-1 infection in THP-1 macrophages and MDMs. (A) TNF-α and (B) IL-6 cytokine induction in naïve, HIV-BaL-infected, and/or stimulated with HCV core, and <t>β-galactosidase</t> protein control was measured by ELISA. (C) THP-1 macrophages and (D) MDMs were infected with HIV-BaL for 12 hours. Cells were left unstimulated or treated with HCV core, or <t>β-galactosidase</t> protein control, in the presence or absence of specific human monoclonal nAbs anti-TNF-α and/or anti-IL-6. HIV infectivity was determined by luciferase activity in cell lysates 48 hours after infection. Data are shown as relative fold induction of HIV infectivity (mean ± SD). Results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. *Significantly different ( P
    β Galactosidase β Gal Control Protein, supplied by Virogen, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega mouse β galactosidase β gal
    HCV core-induced TNF-α and IL-6 mediate the enhancement of HIV-1 infection in THP-1 macrophages and MDMs. (A) TNF-α and (B) IL-6 cytokine induction in naïve, HIV-BaL-infected, and/or stimulated with HCV core, and <t>β-galactosidase</t> protein control was measured by ELISA. (C) THP-1 macrophages and (D) MDMs were infected with HIV-BaL for 12 hours. Cells were left unstimulated or treated with HCV core, or <t>β-galactosidase</t> protein control, in the presence or absence of specific human monoclonal nAbs anti-TNF-α and/or anti-IL-6. HIV infectivity was determined by luciferase activity in cell lysates 48 hours after infection. Data are shown as relative fold induction of HIV infectivity (mean ± SD). Results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. *Significantly different ( P
    Mouse β Galactosidase β Gal, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson β galactosidase β gal reporter plasmid
    HCV core-induced TNF-α and IL-6 mediate the enhancement of HIV-1 infection in THP-1 macrophages and MDMs. (A) TNF-α and (B) IL-6 cytokine induction in naïve, HIV-BaL-infected, and/or stimulated with HCV core, and <t>β-galactosidase</t> protein control was measured by ELISA. (C) THP-1 macrophages and (D) MDMs were infected with HIV-BaL for 12 hours. Cells were left unstimulated or treated with HCV core, or <t>β-galactosidase</t> protein control, in the presence or absence of specific human monoclonal nAbs anti-TNF-α and/or anti-IL-6. HIV infectivity was determined by luciferase activity in cell lysates 48 hours after infection. Data are shown as relative fold induction of HIV infectivity (mean ± SD). Results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. *Significantly different ( P
    β Galactosidase β Gal Reporter Plasmid, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β galactosidase β gal reporter plasmid/product/Becton Dickinson
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    92
    Cell Signaling Technology Inc β galactosidase β gal staining kit
    HCV core-induced TNF-α and IL-6 mediate the enhancement of HIV-1 infection in THP-1 macrophages and MDMs. (A) TNF-α and (B) IL-6 cytokine induction in naïve, HIV-BaL-infected, and/or stimulated with HCV core, and <t>β-galactosidase</t> protein control was measured by ELISA. (C) THP-1 macrophages and (D) MDMs were infected with HIV-BaL for 12 hours. Cells were left unstimulated or treated with HCV core, or <t>β-galactosidase</t> protein control, in the presence or absence of specific human monoclonal nAbs anti-TNF-α and/or anti-IL-6. HIV infectivity was determined by luciferase activity in cell lysates 48 hours after infection. Data are shown as relative fold induction of HIV infectivity (mean ± SD). Results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. *Significantly different ( P
    β Galactosidase β Gal Staining Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β galactosidase β gal staining kit/product/Cell Signaling Technology Inc
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    Image Search Results


    The effect of Box A, Box C and Box B mutations on origin-dependent DNA replication and flanking gene expression in the context of VZV-MSP superinfection. A) A typical Southern blot analysis of the effects on DNA replication of CGC motif mutation in Box A, C and B to AAA. The upper band (R) indicates the position of DpnI resistant DNA resulting from replication in MeWo cells. The lower band (U) indicates the position of unreplicated input plasmid. B) A histogram summarizing the data from three independent DpnI replication assays analyzed at 48 hrs post VZV-MSP superinfection. C) Results of triplicate assays comparing the effect of the presence of Box A, Box C and Box B mutations on the expression levels of the Renilla luciferase reporter gene present at the position of ORF62 and D) the firefly luciferase reporter gene present at the position of ORF63. The promoter activities in the presence of VZV-MSP superinfection are represented as RLU/β-Gal units. The black and grey bars represent experiments done in the absence and presence of 400 μg/ml of PAA respectively. Statistical significance was determined by a oneway ANOVA analysis of variance followed by Tukey's post hoc test.

    Journal: Virology

    Article Title: Varicella-Zoster Virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription

    doi: 10.1016/j.virol.2015.02.049

    Figure Lengend Snippet: The effect of Box A, Box C and Box B mutations on origin-dependent DNA replication and flanking gene expression in the context of VZV-MSP superinfection. A) A typical Southern blot analysis of the effects on DNA replication of CGC motif mutation in Box A, C and B to AAA. The upper band (R) indicates the position of DpnI resistant DNA resulting from replication in MeWo cells. The lower band (U) indicates the position of unreplicated input plasmid. B) A histogram summarizing the data from three independent DpnI replication assays analyzed at 48 hrs post VZV-MSP superinfection. C) Results of triplicate assays comparing the effect of the presence of Box A, Box C and Box B mutations on the expression levels of the Renilla luciferase reporter gene present at the position of ORF62 and D) the firefly luciferase reporter gene present at the position of ORF63. The promoter activities in the presence of VZV-MSP superinfection are represented as RLU/β-Gal units. The black and grey bars represent experiments done in the absence and presence of 400 μg/ml of PAA respectively. Statistical significance was determined by a oneway ANOVA analysis of variance followed by Tukey's post hoc test.

    Article Snippet: Cells were transfected with one μg of each reporter vector (pLitmus R62/63F) using Lipofectamine reagent (Invitrogen, Carlsbad, CA), along with 0.4 μg of β-galactosidase (β-Gal)-expressing plasmid (Invitrogen, Carlsbad, CA) as a control of transfection efficiency.

    Techniques: Expressing, Southern Blot, Mutagenesis, Plasmid Preparation, Luciferase

    (A) A minimal domain of NS3 required for interaction was defined using the yeast two-hybrid assay. N- and C-terminal truncations in NS3 were made, and the fragments were cloned into pAS2–1. These were transformed into PJ69–2A, and transformants were mated with Y187 carrying pACT-NS3. The resulting diploids were patched onto −Trp −Leu plates, replica plated onto −His and −Ade plates, and also lifted onto a nylon membrane for the β-Gal assay. A fragment encompassing aa 182 to 335 near the N terminus of the helicase domain, which includes conserved residues important for NTP-binding, NTPase, and helicase activities, was defined to be the minimal region required for interaction with NS3. Positive interactions were indicated by growth on −His and −Ade plates and by the presence of β-Gal activity. (B) IP between Flag-NS3 and myc-tagged NS3 proteins. COS cells were transfected with Flag-NS3 and myc-NS3 or with myc-NS3 alone. Total protein (100 μg) from transfected cells was used for IP using an anti-Flag agarose gel. Lane 1, myc-NS3 was detected in the complex precipitated with an anti-Flag gel, demonstrating interaction between myc-NS3 and Flag-NS3. Lane 2, myc-NS3 was not precipitated by the anti-Flag gel in the absence of Flag-NS3, showing the specificity of the anti-Flag gel used for IP. Lanes 3 and 4, 1/10 of the input IP proteins was probed with anti-Flag antibody to show that Flag-NS3 was specifically precipitated by the anti-Flag gel. Lanes 5 through 7, Western blot of total cell lysate (20 μg loaded) to detect the expression of each of the tagged NS3 proteins in transfected cells. IgG, immunoglobulin G.

    Journal: Journal of Virology

    Article Title: Mutations That Affect Dimer Formation and Helicase Activity of the Hepatitis C Virus Helicase

    doi: 10.1128/JVI.75.1.205-214.2001

    Figure Lengend Snippet: (A) A minimal domain of NS3 required for interaction was defined using the yeast two-hybrid assay. N- and C-terminal truncations in NS3 were made, and the fragments were cloned into pAS2–1. These were transformed into PJ69–2A, and transformants were mated with Y187 carrying pACT-NS3. The resulting diploids were patched onto −Trp −Leu plates, replica plated onto −His and −Ade plates, and also lifted onto a nylon membrane for the β-Gal assay. A fragment encompassing aa 182 to 335 near the N terminus of the helicase domain, which includes conserved residues important for NTP-binding, NTPase, and helicase activities, was defined to be the minimal region required for interaction with NS3. Positive interactions were indicated by growth on −His and −Ade plates and by the presence of β-Gal activity. (B) IP between Flag-NS3 and myc-tagged NS3 proteins. COS cells were transfected with Flag-NS3 and myc-NS3 or with myc-NS3 alone. Total protein (100 μg) from transfected cells was used for IP using an anti-Flag agarose gel. Lane 1, myc-NS3 was detected in the complex precipitated with an anti-Flag gel, demonstrating interaction between myc-NS3 and Flag-NS3. Lane 2, myc-NS3 was not precipitated by the anti-Flag gel in the absence of Flag-NS3, showing the specificity of the anti-Flag gel used for IP. Lanes 3 and 4, 1/10 of the input IP proteins was probed with anti-Flag antibody to show that Flag-NS3 was specifically precipitated by the anti-Flag gel. Lanes 5 through 7, Western blot of total cell lysate (20 μg loaded) to detect the expression of each of the tagged NS3 proteins in transfected cells. IgG, immunoglobulin G.

    Article Snippet: In this system, the presence of an interaction between two viral proteins was indicated by the activation of the reporter genes HIS3 and ADE2 , which allow for growth on −His and −Ade media, respectively, and LacZ, a β-galactosidase (β-Gal) that produces a blue color when the substrate X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (Sigma) is cleaved.

    Techniques: Y2H Assay, Clone Assay, Transformation Assay, β-Gal Assay, Binding Assay, Activity Assay, Transfection, Agarose Gel Electrophoresis, Western Blot, Expressing

    Positions of some of the mutations that disrupted interaction between two minimal regions. Mutations in the minimal fragment resulted in a complete loss of interaction with a wild-type minimal region (min X min). When transferred to a full-length helicase, the mutations resulted in weaker interactions (w) with a wild-type helicase (hel X hel), i.e., His + , Ade − , and β-Gal − , compared to the interaction between two wild-type helicases. The conserved helicase motifs are underlined; mutations selected for in vitro helicase and dimerization assays are in bold.

    Journal: Journal of Virology

    Article Title: Mutations That Affect Dimer Formation and Helicase Activity of the Hepatitis C Virus Helicase

    doi: 10.1128/JVI.75.1.205-214.2001

    Figure Lengend Snippet: Positions of some of the mutations that disrupted interaction between two minimal regions. Mutations in the minimal fragment resulted in a complete loss of interaction with a wild-type minimal region (min X min). When transferred to a full-length helicase, the mutations resulted in weaker interactions (w) with a wild-type helicase (hel X hel), i.e., His + , Ade − , and β-Gal − , compared to the interaction between two wild-type helicases. The conserved helicase motifs are underlined; mutations selected for in vitro helicase and dimerization assays are in bold.

    Article Snippet: In this system, the presence of an interaction between two viral proteins was indicated by the activation of the reporter genes HIS3 and ADE2 , which allow for growth on −His and −Ade media, respectively, and LacZ, a β-galactosidase (β-Gal) that produces a blue color when the substrate X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (Sigma) is cleaved.

    Techniques: In Vitro

    (A) CNF1 subcellular localization and secretion in E. coli RS218. Western blot analysis was carried out with the cytoplasmic (C) and periplasmic (P) fractions from RS218, Δ fdx , and CΔ fdx (with 0.1% arabinose to promote the transcription of complemented fdx gene). The amount of cytoplasmic protein loaded was 40 μg, and the amount of periplasmic protein loaded was equal to the total periplasmic protein that was collected from 3 ×10 9 bacteria (the number of bacteria was estimated from the optical density at 620 nm [OD 620 ]). CNF1, PhoA, β-Gal, and DsbA were detected by their respective specific antibodies, as described in Materials and Methods. (B) SDS-PAGE analysis of the protein profile in the cytoplasmic and periplasmic fractions prepared from RS218 and Δ fdx as indicated in the figure. M, molecular marker; size positions are indicated along with the masses on the left. The amount of cytoplasmic protein loaded was 15 μg. The loaded periplasmic protein was equal to the total periplasmic protein that was collected from 10 10 bacteria cells (the number of bacteria was estimated from the OD 620 ).

    Journal: Infection and Immunity

    Article Title: Ferredoxin Is Involved in Secretion of Cytotoxic Necrotizing Factor 1 across the Cytoplasmic Membrane in Escherichia coli K1 ▿

    doi: 10.1128/IAI.00674-09

    Figure Lengend Snippet: (A) CNF1 subcellular localization and secretion in E. coli RS218. Western blot analysis was carried out with the cytoplasmic (C) and periplasmic (P) fractions from RS218, Δ fdx , and CΔ fdx (with 0.1% arabinose to promote the transcription of complemented fdx gene). The amount of cytoplasmic protein loaded was 40 μg, and the amount of periplasmic protein loaded was equal to the total periplasmic protein that was collected from 3 ×10 9 bacteria (the number of bacteria was estimated from the optical density at 620 nm [OD 620 ]). CNF1, PhoA, β-Gal, and DsbA were detected by their respective specific antibodies, as described in Materials and Methods. (B) SDS-PAGE analysis of the protein profile in the cytoplasmic and periplasmic fractions prepared from RS218 and Δ fdx as indicated in the figure. M, molecular marker; size positions are indicated along with the masses on the left. The amount of cytoplasmic protein loaded was 15 μg. The loaded periplasmic protein was equal to the total periplasmic protein that was collected from 10 10 bacteria cells (the number of bacteria was estimated from the OD 620 ).

    Article Snippet: Primary antibodies used in this study were anti-CNF1 monoclonal antibody (DD1) , alkaline phosphatase (PhoA) monoclonal antibody (Millipore), and β-galactosidase (β-Gal) antiserum (Millipore), and disulfide oxidoreductase (DsbA) antiserum was also used ( ).

    Techniques: Western Blot, SDS Page, Marker

    A large portion of neurons infected in caudal VTA project to the NAc and are dopaminergic. The number of β-gal-positive neurons did not significantly differ between rostral and caudal VTA. However, more neurons were retrogradely labeled in caudal VTA than in rostral VTA after injection of HS in to the NAc. More neurons in caudal VTA than in rostral VTA also were colabeled with β-gal and HS. Most of the HS- and β-gal-positive neurons in rostral and caudal VTA were also TH positive. B-G , A representative section from caudal VTA, triple labeled for HS ( B ), β-gal ( C ), and TH ( D ). E , Merged image from B and C showing neurons colabeled with HS and β-gal. F , Merged image from B and D showing neurons colabeled with HS and TH. G , Merged image from B-D showing triple-labeled neurons (arrows). Data are expressed as cell numbers per section ± SEM ( n = 3). * p

    Journal: The Journal of Neuroscience

    Article Title: Regulation of Drug Reward by cAMP Response Element-Binding Protein: Evidence for Two Functionally Distinct Subregions of the Ventral Tegmental Area

    doi: 10.1523/JNEUROSCI.0345-05.2005

    Figure Lengend Snippet: A large portion of neurons infected in caudal VTA project to the NAc and are dopaminergic. The number of β-gal-positive neurons did not significantly differ between rostral and caudal VTA. However, more neurons were retrogradely labeled in caudal VTA than in rostral VTA after injection of HS in to the NAc. More neurons in caudal VTA than in rostral VTA also were colabeled with β-gal and HS. Most of the HS- and β-gal-positive neurons in rostral and caudal VTA were also TH positive. B-G , A representative section from caudal VTA, triple labeled for HS ( B ), β-gal ( C ), and TH ( D ). E , Merged image from B and C showing neurons colabeled with HS and β-gal. F , Merged image from B and D showing neurons colabeled with HS and TH. G , Merged image from B-D showing triple-labeled neurons (arrows). Data are expressed as cell numbers per section ± SEM ( n = 3). * p

    Article Snippet: In experiments using transgenic mice, LacZ expression was detected with a polyclonal antibody to β-galactosidase (β-gal) (1:5000; Biogenesis, Poole, UK), and some sections were double labeled for tyrosine hydroxylase (TH) (1: 1000; Chemicon, Temecula, CA) to determine whether the β-gal-positive neurons were dopaminergic.

    Techniques: Infection, Labeling, Injection

    Increased Ca 2 + influx into and CK efflux from BIO14.6 myotubes and their correction by δ-SG gene transfer. (a) 45 Ca 2+ uptake into normal or BIO14.6 myotubes measured under resting conditions with or without SK F96365 (SK F), ruthenium red (RR), or Nifedipine. The Gd 3+ -inhibitable fractions are shown. (b) External Ca 2+ -induced changes in fluo-4 fluorescence in normal or BIO14.6 myotubes. The bar graph shows the maximal increments of fluorescence in myotubes pretreated with or without 50 μM SK F96365. ΔF/F 0 is the ratio between the fluorescence increment and the fluorescence before Ca 2+ addition. Numbers in parentheses correspond to the number of cells studied. (c) CK efflux from BIO14.6 myotubes subjected to cyclic stretch under indicated conditions. (d) Immunoblot assay and immunohistochemistry (IH) of BIO14.6 myotubes infected with Ad.β-gal or Ad.δ-SG. (e and f) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch–induced CK efflux in BIO14.6 myotubes insfected with Ad.β-gal or Ad.δ-SG. TG, thapsigargin (1 μM). Other conditions were similar to b and c. In these panels, error bars show means ± SD and asterisks show P

    Journal: The Journal of Cell Biology

    Article Title: A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel

    doi: 10.1083/jcb.200301101

    Figure Lengend Snippet: Increased Ca 2 + influx into and CK efflux from BIO14.6 myotubes and their correction by δ-SG gene transfer. (a) 45 Ca 2+ uptake into normal or BIO14.6 myotubes measured under resting conditions with or without SK F96365 (SK F), ruthenium red (RR), or Nifedipine. The Gd 3+ -inhibitable fractions are shown. (b) External Ca 2+ -induced changes in fluo-4 fluorescence in normal or BIO14.6 myotubes. The bar graph shows the maximal increments of fluorescence in myotubes pretreated with or without 50 μM SK F96365. ΔF/F 0 is the ratio between the fluorescence increment and the fluorescence before Ca 2+ addition. Numbers in parentheses correspond to the number of cells studied. (c) CK efflux from BIO14.6 myotubes subjected to cyclic stretch under indicated conditions. (d) Immunoblot assay and immunohistochemistry (IH) of BIO14.6 myotubes infected with Ad.β-gal or Ad.δ-SG. (e and f) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch–induced CK efflux in BIO14.6 myotubes insfected with Ad.β-gal or Ad.δ-SG. TG, thapsigargin (1 μM). Other conditions were similar to b and c. In these panels, error bars show means ± SD and asterisks show P

    Article Snippet: For adenoviral gene transfer, we ligated full-length cDNA of hamster δ-SG or β-galactosidase (β-gal), or full-length antisense cDNA of mouse GRC into the Adeno-XTM viral vector (CLONTECH Laboratories, Inc.) according to the manufacturer's protocol.

    Techniques: Fluorescence, Immunohistochemistry, Infection

    Effect of GRC antisense expression on BIO14.6 myotubes. (a) Immunoblot assay for GRC and β-dystroglycan (β-DG; top) and GRC immunohistochemistry (middle) of BIO14.6 myotubes infected with Ad.β-gal or Ad-antisense–GRC cDNA (Ad.asGRC). Cell surface GRC levels were estimated by labeling antisense-treated or nontreated myotubes with NHS-biotin and by further analyzing streptavidin agarose-bound (B) and -unbound (U) fractions by immunoblot assay with anti-GRC (bottom). (b and c) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch-induced CK efflux from antisense-treated or nontreated myotubes. Other conditions were the same as those in Fig. 3 (b and c). Error bars show means ± SD and asterisks show P

    Journal: The Journal of Cell Biology

    Article Title: A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel

    doi: 10.1083/jcb.200301101

    Figure Lengend Snippet: Effect of GRC antisense expression on BIO14.6 myotubes. (a) Immunoblot assay for GRC and β-dystroglycan (β-DG; top) and GRC immunohistochemistry (middle) of BIO14.6 myotubes infected with Ad.β-gal or Ad-antisense–GRC cDNA (Ad.asGRC). Cell surface GRC levels were estimated by labeling antisense-treated or nontreated myotubes with NHS-biotin and by further analyzing streptavidin agarose-bound (B) and -unbound (U) fractions by immunoblot assay with anti-GRC (bottom). (b and c) External Ca 2+ -induced changes in fluo-4 fluorescence and cyclic stretch-induced CK efflux from antisense-treated or nontreated myotubes. Other conditions were the same as those in Fig. 3 (b and c). Error bars show means ± SD and asterisks show P

    Article Snippet: For adenoviral gene transfer, we ligated full-length cDNA of hamster δ-SG or β-galactosidase (β-gal), or full-length antisense cDNA of mouse GRC into the Adeno-XTM viral vector (CLONTECH Laboratories, Inc.) according to the manufacturer's protocol.

    Techniques: Expressing, Immunohistochemistry, Infection, Labeling, Fluorescence

    miR-218-5p directly downregulates EGFR in NSCLC cells A. Schematic representation of the putative miR-218-5p binding sequence on the 3′-UTR of the EGFR mRNA. B. The luciferase reporter plasmid encoding the full-length 3′-UTR of EGFR was co-transfected into the A549 and H1975 cells along with miR-218-5p mimic or scrambled mimic. The luciferase activities were normalized to the β-galactosidase levels of the control (**P

    Journal: Oncotarget

    Article Title: Tumor-suppressive miR-218-5p inhibits cancer cell proliferation and migration via EGFR in non-small cell lung cancer

    doi: 10.18632/oncotarget.8576

    Figure Lengend Snippet: miR-218-5p directly downregulates EGFR in NSCLC cells A. Schematic representation of the putative miR-218-5p binding sequence on the 3′-UTR of the EGFR mRNA. B. The luciferase reporter plasmid encoding the full-length 3′-UTR of EGFR was co-transfected into the A549 and H1975 cells along with miR-218-5p mimic or scrambled mimic. The luciferase activities were normalized to the β-galactosidase levels of the control (**P

    Article Snippet: The cells were cultured in 12-well plates, and each well was transfected with 0.8 μg of firefly luciferase reporter plasmid, 0.8 μg of a β-galactosidase (β-gal) expression plasmid (Ambion), and equal amounts (40 pmol) of miR-218-5p mimic or scrambled mimic using Lipofectamine 2000 (Invitrogen).

    Techniques: Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Transfection

    ( A ) Extents of expression in COS-7 cell cultures of the β-gal gene mediated in the presence of serum by PEI2 (bars a–c) and PEI2–GNPII (bars d–f) at different N/P ratios. Bars a and d, N/P = 90; bars b and e, N/P = 120; bars c and f, N/P = 150. ( B ) Extents of expression in COS-7 cell cultures of the β-gal gene mediated in the presence of serum by PEI25 (bar a), dodecyl–PEI2 (bars b and c), PEI2–GNPII (bar d), and PEI2–GNPII + dodecyl–PEI2 (bars e and f). Bar a, N/P = 10; bar b, N/P = 20; bar c, N/P = 40; bar d, N/P = 150; bar e, N/P = 150 + 20; bar f, N/P = 150 + 40. Cells transfected with the plasmid in the absence of polycations showed no appreciable β-gal activity.

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

    Article Title: Conjugation to gold nanoparticles enhances polyethylenimine's transfer of plasmid DNA into mammalian cells

    doi: 10.1073/pnas.1233634100

    Figure Lengend Snippet: ( A ) Extents of expression in COS-7 cell cultures of the β-gal gene mediated in the presence of serum by PEI2 (bars a–c) and PEI2–GNPII (bars d–f) at different N/P ratios. Bars a and d, N/P = 90; bars b and e, N/P = 120; bars c and f, N/P = 150. ( B ) Extents of expression in COS-7 cell cultures of the β-gal gene mediated in the presence of serum by PEI25 (bar a), dodecyl–PEI2 (bars b and c), PEI2–GNPII (bar d), and PEI2–GNPII + dodecyl–PEI2 (bars e and f). Bar a, N/P = 10; bar b, N/P = 20; bar c, N/P = 40; bar d, N/P = 150; bar e, N/P = 150 + 20; bar f, N/P = 150 + 40. Cells transfected with the plasmid in the absence of polycations showed no appreciable β-gal activity.

    Article Snippet: Plasmid. gWiz Beta-Gal (8,278 bp) encoding the β-galactosidase (β-gal) gene was purchased from Aldevron (Fargo, ND).

    Techniques: Expressing, Transfection, Plasmid Preparation, Activity Assay

    NS1 containing cell culture supernatants inhibit the TLR3 response in HeLa cells. (A) HeLa cells were transfected with an IFNβ-Luc reporter construct and a β-gal expressing plasmid and subsequently co-cultured with either regular HeLa cells or HeLa cells harboring a WNV replicon (HeLa rep). Luc activity was determined 4h after the addition of pIC to the culture media and normalized to β-gal activity. Asterisks represent statistically significant differences, determined by student’s t-test, comparing the pIC response of HeLa:HeLa rep samples to that of HeLa:HeLa samples. (B) HeLa cells transfected with IFNβ reporter and β-gal as above were incubated for 16h with cell culture supernatants from either regular HeLa cells or HeLa rep cells then stimulated with pIC before Luc activity was determined. Asterisks represent statistically significant differences, determined by student’s t-test comparing the pIC response of HeLa rep cells to that of HeLa cells. (C) HeLa cells were incubated with cell culture supernatants from either 293T-Ctrl or 293T-NS1 cells for 16h. NS1 association with the cells was detected by immunofluorescence of permeabilized cells using WNV MHIAF. (D) HeLa cells were incubated with control or sNS1-containing supernatants for 16h and extensively washed before lysis. Whole cell lysates were analyzed by immunoblot with WNV MHIAF followed by HRP-labeled mouse antibody. (E) NS1 association with naïve cells by flow cytometry. HeLa cells were incubated with control or sNS1-containing supernatants for 6h and 24h either left unpermeabilized to detect NS1 on the cell surface or permeabilized prior to staining to detect internalized NS1. NS1 was detected with an NS1-specific antibody. (F) Secreted NS1-containing cell culture supernatants inhibit the TLR3 response in HeLa cells. HeLa cells transfected with IFNβ reporter construct and β-gal were incubated with supernatants from either 293T-Ctrl or 293T-NS1 cells for 16h and stimulated as above. Asterisks represent statistically significant differences, determined by student’s t-test of sNS1-containing supernatant treated samples compared to control treated samples

    Journal: Virology

    Article Title: Modulation of innate immune signaling by the secreted form of the West Nile virus NS1 glycoprotein

    doi: 10.1016/j.virol.2014.04.036

    Figure Lengend Snippet: NS1 containing cell culture supernatants inhibit the TLR3 response in HeLa cells. (A) HeLa cells were transfected with an IFNβ-Luc reporter construct and a β-gal expressing plasmid and subsequently co-cultured with either regular HeLa cells or HeLa cells harboring a WNV replicon (HeLa rep). Luc activity was determined 4h after the addition of pIC to the culture media and normalized to β-gal activity. Asterisks represent statistically significant differences, determined by student’s t-test, comparing the pIC response of HeLa:HeLa rep samples to that of HeLa:HeLa samples. (B) HeLa cells transfected with IFNβ reporter and β-gal as above were incubated for 16h with cell culture supernatants from either regular HeLa cells or HeLa rep cells then stimulated with pIC before Luc activity was determined. Asterisks represent statistically significant differences, determined by student’s t-test comparing the pIC response of HeLa rep cells to that of HeLa cells. (C) HeLa cells were incubated with cell culture supernatants from either 293T-Ctrl or 293T-NS1 cells for 16h. NS1 association with the cells was detected by immunofluorescence of permeabilized cells using WNV MHIAF. (D) HeLa cells were incubated with control or sNS1-containing supernatants for 16h and extensively washed before lysis. Whole cell lysates were analyzed by immunoblot with WNV MHIAF followed by HRP-labeled mouse antibody. (E) NS1 association with naïve cells by flow cytometry. HeLa cells were incubated with control or sNS1-containing supernatants for 6h and 24h either left unpermeabilized to detect NS1 on the cell surface or permeabilized prior to staining to detect internalized NS1. NS1 was detected with an NS1-specific antibody. (F) Secreted NS1-containing cell culture supernatants inhibit the TLR3 response in HeLa cells. HeLa cells transfected with IFNβ reporter construct and β-gal were incubated with supernatants from either 293T-Ctrl or 293T-NS1 cells for 16h and stimulated as above. Asterisks represent statistically significant differences, determined by student’s t-test of sNS1-containing supernatant treated samples compared to control treated samples

    Article Snippet: Briefly, 150ng each of IFN-β pGL3 and pCMV β-galactosidase (β-Gal) (Invitrogen) were co-transfected into HeLa cells, using the TransIT-LT1transfection reagent (Mirus).

    Techniques: Cell Culture, Transfection, Construct, Expressing, Plasmid Preparation, Activity Assay, Incubation, Immunofluorescence, Lysis, Labeling, Flow Cytometry, Cytometry, Staining

    HSV-1 entry into nTERT cells requires the gD receptor nectin-1. (A and B) Nectin-1 (A) or HVEM (B) mRNA was quantitated in HeLa and nTERT cells by qRT-PCR using the ΔΔ C T method. The housekeeping gene RPLP0 was used to normalize expression of both genes. (C and D) nTERT or HeLa cells were reverse transfected with 30 nM nectin-1, HVEM, or control siRNAs. Seventy-two hours later, cell RNA was extracted from half of the siRNA-transfected cells, and nectin-1 and HVEM mRNA levels were quantitated by qRT-PCR using the ΔΔ C T method. (C) The housekeeping gene RPLP0 was used to normalize expression of both genes. The remaining cells were transferred in triplicate to 96-well plates and incubated for a further 16 h. These cells were infected on ice with 110lacZ virus at a multiplicity of infection of 2 for 1 h prior to replacing the inoculum with fresh media at 37°C and incubating for a further 4 h. Cells were lysed and relative β-galactosidase activity measured. (D) A representative experiment is shown as the means ± standard errors of the data relative to the negative siRNA control.

    Journal: Journal of Virology

    Article Title: Herpes Simplex Virus 1 Enters Human Keratinocytes by a Nectin-1-Dependent, Rapid Plasma Membrane Fusion Pathway That Functions at Low Temperature

    doi: 10.1128/JVI.01582-16

    Figure Lengend Snippet: HSV-1 entry into nTERT cells requires the gD receptor nectin-1. (A and B) Nectin-1 (A) or HVEM (B) mRNA was quantitated in HeLa and nTERT cells by qRT-PCR using the ΔΔ C T method. The housekeeping gene RPLP0 was used to normalize expression of both genes. (C and D) nTERT or HeLa cells were reverse transfected with 30 nM nectin-1, HVEM, or control siRNAs. Seventy-two hours later, cell RNA was extracted from half of the siRNA-transfected cells, and nectin-1 and HVEM mRNA levels were quantitated by qRT-PCR using the ΔΔ C T method. (C) The housekeeping gene RPLP0 was used to normalize expression of both genes. The remaining cells were transferred in triplicate to 96-well plates and incubated for a further 16 h. These cells were infected on ice with 110lacZ virus at a multiplicity of infection of 2 for 1 h prior to replacing the inoculum with fresh media at 37°C and incubating for a further 4 h. Cells were lysed and relative β-galactosidase activity measured. (D) A representative experiment is shown as the means ± standard errors of the data relative to the negative siRNA control.

    Article Snippet: The medium was removed by aspiration and the cells were washed twice with ice-cold PBS before being lysed and analyzed using the Invitrogen β-galactosidase (β-gal) assay kit.

    Techniques: Quantitative RT-PCR, Expressing, Transfection, Incubation, Infection, Activity Assay

    Role of MLK3 and JNK1 pathways on G17-induced MMP7 transcription. A, Subconfluent AGSE cells were transiently transfected with the MMP7-luciferase vector along with a β-Gal vector (for normalization of transfection). Forty-eight hours post-transfection,

    Journal: Molecular Endocrinology

    Article Title: Mixed Lineage Kinase-3/JNK1 Axis Promotes Migration of Human Gastric Cancer Cells following Gastrin Stimulation

    doi: 10.1210/me.2009-0387

    Figure Lengend Snippet: Role of MLK3 and JNK1 pathways on G17-induced MMP7 transcription. A, Subconfluent AGSE cells were transiently transfected with the MMP7-luciferase vector along with a β-Gal vector (for normalization of transfection). Forty-eight hours post-transfection,

    Article Snippet: DMEM, LipofectAMINE 2000, and β-galactosidase (β-Gal) assay kit were purchased from Invitrogen (Carlsbad, CA); amidated gastrin (G17) was from Bachem (King of Prussia, PA), and the luciferase assay kit was from Promega Corp. (Madison, WI).

    Techniques: Transfection, Luciferase, Plasmid Preparation

    Role of c-Jun on G17-induced MMP7 transcription. A, Subconfluent AGSE cells were transiently transfected with the MMP7-luciferase and β-Gal vectors along with either control siRNA (lanes 1and 2) or c-Jun siRNA (lanes 3 and 4). Luciferase and β-Gal

    Journal: Molecular Endocrinology

    Article Title: Mixed Lineage Kinase-3/JNK1 Axis Promotes Migration of Human Gastric Cancer Cells following Gastrin Stimulation

    doi: 10.1210/me.2009-0387

    Figure Lengend Snippet: Role of c-Jun on G17-induced MMP7 transcription. A, Subconfluent AGSE cells were transiently transfected with the MMP7-luciferase and β-Gal vectors along with either control siRNA (lanes 1and 2) or c-Jun siRNA (lanes 3 and 4). Luciferase and β-Gal

    Article Snippet: DMEM, LipofectAMINE 2000, and β-galactosidase (β-Gal) assay kit were purchased from Invitrogen (Carlsbad, CA); amidated gastrin (G17) was from Bachem (King of Prussia, PA), and the luciferase assay kit was from Promega Corp. (Madison, WI).

    Techniques: Transfection, Luciferase

    Plasmids used for the promoter assays. The CELO virus MLP sequence is between nt 7300 and 7530. The ORF 1 promoter (PORF1) sequence is between nt 204 and 745. These two sequences were amplified by PCR and cloned upstream of the β-galactosidase gene from pCMVβGal.

    Journal: Journal of Virology

    Article Title: Transcriptional Organization of the Avian Adenovirus CELO

    doi:

    Figure Lengend Snippet: Plasmids used for the promoter assays. The CELO virus MLP sequence is between nt 7300 and 7530. The ORF 1 promoter (PORF1) sequence is between nt 204 and 745. These two sequences were amplified by PCR and cloned upstream of the β-galactosidase gene from pCMVβGal.

    Article Snippet: The β-Gal assay kit from Invitrogen provides the reagents required to measure the levels of active β-galactosidase expressed on cells transfected with plasmids expressing lacZ .

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay

    β-Galactosidase assay on LMH cells. Cells were transfected with plasmid pBlueScript KS(+)/PORF1/βGal. The results were observed 48 h later.

    Journal: Journal of Virology

    Article Title: Transcriptional Organization of the Avian Adenovirus CELO

    doi:

    Figure Lengend Snippet: β-Galactosidase assay on LMH cells. Cells were transfected with plasmid pBlueScript KS(+)/PORF1/βGal. The results were observed 48 h later.

    Article Snippet: The β-Gal assay kit from Invitrogen provides the reagents required to measure the levels of active β-galactosidase expressed on cells transfected with plasmids expressing lacZ .

    Techniques: Transfection, Plasmid Preparation

    Activation of HIV-1 gene expression by Tat. HeLa cells were cotransfected with the reporter constructs HIV-1 LTR-CAT and pCH110 (β-Gal) together with plasmids (pDex) that expressed either the β-globin gene (bar 1), the wild type tat gene (bar 2), or the mutated tat genes corresponding to [E2G, D5G, E9G] (bar 3), P3L (bar 4), P[6, 10]L (bar 5), P[10, 14]L (bar 6), C27S (bar 7), K41A (bar 8), and K/R[50-57]G (bar 9). The cells were harvested at 48 h posttransfection, and equal amounts of protein were normalized to β-Gal activity and assayed for CAT protein by ELISA. The transfections were performed three times with the standard deviations indicated.

    Journal: Journal of Virology

    Article Title: Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †

    doi:

    Figure Lengend Snippet: Activation of HIV-1 gene expression by Tat. HeLa cells were cotransfected with the reporter constructs HIV-1 LTR-CAT and pCH110 (β-Gal) together with plasmids (pDex) that expressed either the β-globin gene (bar 1), the wild type tat gene (bar 2), or the mutated tat genes corresponding to [E2G, D5G, E9G] (bar 3), P3L (bar 4), P[6, 10]L (bar 5), P[10, 14]L (bar 6), C27S (bar 7), K41A (bar 8), and K/R[50-57]G (bar 9). The cells were harvested at 48 h posttransfection, and equal amounts of protein were normalized to β-Gal activity and assayed for CAT protein by ELISA. The transfections were performed three times with the standard deviations indicated.

    Article Snippet: Plasmid pCH110, which expressed the β-galactosidase (β-Gal) gene, was obtained from Amersham Pharmacia Biotech.

    Techniques: Activation Assay, Expressing, Construct, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection

    Transactivation of PPARγ 1 /RXRγ heterodimers via E- and H-FABP-PPRE without and with ligand ( A ) HepG2 cells were transiently co-transfected with pCAT3 promoter plasmid containing the PPRE, β-Gal, without and with plasmids for PPARγ 1 and RXRγ. After 42 h, cells were harvested, and β-Gal and CAT concentrations were determined by ELISAs. For each PPRE, experiments were normalized to those without ectopic PPARγ 1 /RXRγ, set to unity. Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6). ( B ) HepG2 cells were transiently co-transfected with pCAT3 promoter plasmid containing the PPRE and plasmids for β-Gal, PPARγ 1 and RXRγ. DMSO and DMSO plus bezafibrate respectively were added to the medium 4 h after transfection (final concentrations 1% DMSO and 100 μM bezafibrate) and cells were incubated for a further 38 h. After harvest, β-Gal and CAT concentrations were determined by ELISAs. For each PPRE, experiments with ligand were normalized to those with DMSO alone, set to unity. Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6).

    Journal: Biochemical Journal

    Article Title: Functional analysis of peroxisome-proliferator-responsive element motifs in genes of fatty acid-binding proteins

    doi: 10.1042/BJ20031340

    Figure Lengend Snippet: Transactivation of PPARγ 1 /RXRγ heterodimers via E- and H-FABP-PPRE without and with ligand ( A ) HepG2 cells were transiently co-transfected with pCAT3 promoter plasmid containing the PPRE, β-Gal, without and with plasmids for PPARγ 1 and RXRγ. After 42 h, cells were harvested, and β-Gal and CAT concentrations were determined by ELISAs. For each PPRE, experiments were normalized to those without ectopic PPARγ 1 /RXRγ, set to unity. Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6). ( B ) HepG2 cells were transiently co-transfected with pCAT3 promoter plasmid containing the PPRE and plasmids for β-Gal, PPARγ 1 and RXRγ. DMSO and DMSO plus bezafibrate respectively were added to the medium 4 h after transfection (final concentrations 1% DMSO and 100 μM bezafibrate) and cells were incubated for a further 38 h. After harvest, β-Gal and CAT concentrations were determined by ELISAs. For each PPRE, experiments with ligand were normalized to those with DMSO alone, set to unity. Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6).

    Article Snippet: Cells were co-transfected with the aid of Fugene6 transfection reagent (Roche Diagnostics) with expression plasmids of respective PPAR/RXR combinations (1.5 μg/well each), the pCAT3 promoter vector (1.5 μg; Promega) containing either ideal-PPRE (positive control), L-, A-, E- or H-FABP-PPREs (Table ) and pSV-β-galactosidase (β-Gal) (Promega) as internal reference (0.5 μg/well).

    Techniques: Transfection, Plasmid Preparation, Incubation

    Transactivation of Fabpe and Fabph promoter fragments in C 2 C 12 myoblasts affected by bezafibrate C 2 C 12 myoblasts were transfected with pCAT promoter containing one copy of ideal-PPRE (positive control) or with pCAT promoter alone (negative control). In the tests, proper cells were transfected with pCAT promoter preceded by Fabpe 2447 and Fabph 1514 promotor fragments containing the putative PPRE or Fabpe 1716 and Fabph 817 without the proposed PPRE respectively and pSV-β-Gal. Cells were cultured for 48 h after transfection in fresh medium supplemented with 1% DMSO alone as control or with 1% DMSO and 100 μM bezafibrate respectively, and contents of lacZ and cat transcripts were evaluated by Northern blotting. Bars represent the induction of promoter activity in bezafibrate treated cells, normalized to DMSO control. Results are expressed as means±S.D. for four independent experiments.

    Journal: Biochemical Journal

    Article Title: Functional analysis of peroxisome-proliferator-responsive element motifs in genes of fatty acid-binding proteins

    doi: 10.1042/BJ20031340

    Figure Lengend Snippet: Transactivation of Fabpe and Fabph promoter fragments in C 2 C 12 myoblasts affected by bezafibrate C 2 C 12 myoblasts were transfected with pCAT promoter containing one copy of ideal-PPRE (positive control) or with pCAT promoter alone (negative control). In the tests, proper cells were transfected with pCAT promoter preceded by Fabpe 2447 and Fabph 1514 promotor fragments containing the putative PPRE or Fabpe 1716 and Fabph 817 without the proposed PPRE respectively and pSV-β-Gal. Cells were cultured for 48 h after transfection in fresh medium supplemented with 1% DMSO alone as control or with 1% DMSO and 100 μM bezafibrate respectively, and contents of lacZ and cat transcripts were evaluated by Northern blotting. Bars represent the induction of promoter activity in bezafibrate treated cells, normalized to DMSO control. Results are expressed as means±S.D. for four independent experiments.

    Article Snippet: Cells were co-transfected with the aid of Fugene6 transfection reagent (Roche Diagnostics) with expression plasmids of respective PPAR/RXR combinations (1.5 μg/well each), the pCAT3 promoter vector (1.5 μg; Promega) containing either ideal-PPRE (positive control), L-, A-, E- or H-FABP-PPREs (Table ) and pSV-β-galactosidase (β-Gal) (Promega) as internal reference (0.5 μg/well).

    Techniques: Transfection, Positive Control, Negative Control, Cell Culture, Northern Blot, Activity Assay

    Dependence of transactivation potentials of PPAR/RXR heterodimers on FABP-PPREs HepG2 cells were transiently co-transfected with plasmids pSG5-PPARα, -PPARβ, -PPARγ 1 , pcDNA3-PPARγ 2 and pSG5-RXR subtypes respectively together with the pCAT3 promoter plasmid containing a PPRE and pSV-β-Gal. After 42 h, cells were harvested. β-Gal and CAT concentrations were determined by ELISAs; experiments with empty pCAT3 plasmid served as control and were set to unity (neg.). Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6).

    Journal: Biochemical Journal

    Article Title: Functional analysis of peroxisome-proliferator-responsive element motifs in genes of fatty acid-binding proteins

    doi: 10.1042/BJ20031340

    Figure Lengend Snippet: Dependence of transactivation potentials of PPAR/RXR heterodimers on FABP-PPREs HepG2 cells were transiently co-transfected with plasmids pSG5-PPARα, -PPARβ, -PPARγ 1 , pcDNA3-PPARγ 2 and pSG5-RXR subtypes respectively together with the pCAT3 promoter plasmid containing a PPRE and pSV-β-Gal. After 42 h, cells were harvested. β-Gal and CAT concentrations were determined by ELISAs; experiments with empty pCAT3 plasmid served as control and were set to unity (neg.). Results are means±S.D. for two independent experiments, each performed in triplicate ( n =6).

    Article Snippet: Cells were co-transfected with the aid of Fugene6 transfection reagent (Roche Diagnostics) with expression plasmids of respective PPAR/RXR combinations (1.5 μg/well each), the pCAT3 promoter vector (1.5 μg; Promega) containing either ideal-PPRE (positive control), L-, A-, E- or H-FABP-PPREs (Table ) and pSV-β-galactosidase (β-Gal) (Promega) as internal reference (0.5 μg/well).

    Techniques: Transfection, Plasmid Preparation

    Dlx2 -expressing cells and their progeny, defined by Dlx2/tauLacZ (β-gal) expression, intermix with Zebrin II+ VZ cells to form the dorsolateral SVZ. A–C , β-gal (TRITC)- and Zebrin II (FITC)-immunoreactive cells begin to mix perinatally. D–I , Postnatally, Zebrin II+ cells are displaced to form the outer limits of the SVZ, whereas β-gal+ cells constitute the Zebrin II− central region at P6 ( D–F ) and P10 ( G–I ). Note that in A and D , β-gal+ cells also stream into the overlying white matter. Scale bars: B , 100 μm; E , 50 μm; H , 20 μm.

    Journal: The Journal of Neuroscience

    Article Title: Subpallial Dlx2-Expressing Cells Give Rise to Astrocytes and Oligodendrocytes in the Cerebral Cortex and White Matter

    doi: 10.1523/JNEUROSCI.22-22-09821.2002

    Figure Lengend Snippet: Dlx2 -expressing cells and their progeny, defined by Dlx2/tauLacZ (β-gal) expression, intermix with Zebrin II+ VZ cells to form the dorsolateral SVZ. A–C , β-gal (TRITC)- and Zebrin II (FITC)-immunoreactive cells begin to mix perinatally. D–I , Postnatally, Zebrin II+ cells are displaced to form the outer limits of the SVZ, whereas β-gal+ cells constitute the Zebrin II− central region at P6 ( D–F ) and P10 ( G–I ). Note that in A and D , β-gal+ cells also stream into the overlying white matter. Scale bars: B , 100 μm; E , 50 μm; H , 20 μm.

    Article Snippet: Immunohistochemistry with monoclonal antibodies against Zebrin II (1:100; gift from R. Hawkes, University of Calgary, Alberta, Canada), the polysialylated form of neural cell adhesion molecule (PSA-NCAM)/5A5 (1:5; gift from J. Dodd, Columbia University, New York, NY), RC2 (1:1; Developmental Studies Hybridoma Bank, Iowa City, IA), TUJ-1 (1:500; Babco, Richmond, CA), CNPase (1:200; Sigma), and polyclonal antibodies against Dlx2 (1:200; gift from J. Rubenstein, University of California, San Francisco, CA), astrocyte-specific glutamate transporter (GLAST) (1:1000; Chemicon, Temecula, CA), and β-galactosidase (β-gal) (1:1000; Cortex Biochemical, San Leandro, CA) was performed as follows.

    Techniques: Expressing

    Formation of the postnatal SVZ. E10 , Zebrin II is expressed by all cells of the telencephalic primordium. E14 , A Zebrin II+ wedge emerges during corticostriatal sulcus formation. E16, E16 Box , The Zebrin II+ wedge is fenestrated by Dlx2+ or PSA-NCAM+ migratory, subpallium-derived cells. PSA-NCAM+ cells are the progeny of Dlx2+ cells. At P0, the total population of Dlx2-expressing cells and their PSA-NCAM+ progeny can be defined by Dlx2/tauLacZ (β-gal) expression. Subpallium-derived cells defined by Dlx2/tauLacZ (β-gal) expression form the central region of the perinatal SVZ. At P10 (and for the P10 box ), the postnatal SVZ can be delineated into two subpopulations of cells based on the expression of Zebrin II or the Dlx2/tauLacZ reporter. Zebrin II+ residual VZ cells form the outer borders of the SVZ. Cells sharing a Dlx2-expressing subpallial origin, as defined by Dlx2/tauLacZ (β-gal) expression, populate the central SVZ and give rise to astrocytes ( astros ) and oligodendrocytes ( oligos ) in the striatum, white matter, and cortex. Note: model is not drawn to scale.

    Journal: The Journal of Neuroscience

    Article Title: Subpallial Dlx2-Expressing Cells Give Rise to Astrocytes and Oligodendrocytes in the Cerebral Cortex and White Matter

    doi: 10.1523/JNEUROSCI.22-22-09821.2002

    Figure Lengend Snippet: Formation of the postnatal SVZ. E10 , Zebrin II is expressed by all cells of the telencephalic primordium. E14 , A Zebrin II+ wedge emerges during corticostriatal sulcus formation. E16, E16 Box , The Zebrin II+ wedge is fenestrated by Dlx2+ or PSA-NCAM+ migratory, subpallium-derived cells. PSA-NCAM+ cells are the progeny of Dlx2+ cells. At P0, the total population of Dlx2-expressing cells and their PSA-NCAM+ progeny can be defined by Dlx2/tauLacZ (β-gal) expression. Subpallium-derived cells defined by Dlx2/tauLacZ (β-gal) expression form the central region of the perinatal SVZ. At P10 (and for the P10 box ), the postnatal SVZ can be delineated into two subpopulations of cells based on the expression of Zebrin II or the Dlx2/tauLacZ reporter. Zebrin II+ residual VZ cells form the outer borders of the SVZ. Cells sharing a Dlx2-expressing subpallial origin, as defined by Dlx2/tauLacZ (β-gal) expression, populate the central SVZ and give rise to astrocytes ( astros ) and oligodendrocytes ( oligos ) in the striatum, white matter, and cortex. Note: model is not drawn to scale.

    Article Snippet: Immunohistochemistry with monoclonal antibodies against Zebrin II (1:100; gift from R. Hawkes, University of Calgary, Alberta, Canada), the polysialylated form of neural cell adhesion molecule (PSA-NCAM)/5A5 (1:5; gift from J. Dodd, Columbia University, New York, NY), RC2 (1:1; Developmental Studies Hybridoma Bank, Iowa City, IA), TUJ-1 (1:500; Babco, Richmond, CA), CNPase (1:200; Sigma), and polyclonal antibodies against Dlx2 (1:200; gift from J. Rubenstein, University of California, San Francisco, CA), astrocyte-specific glutamate transporter (GLAST) (1:1000; Chemicon, Temecula, CA), and β-galactosidase (β-gal) (1:1000; Cortex Biochemical, San Leandro, CA) was performed as follows.

    Techniques: Derivative Assay, Expressing

    PSA-NCAM+ cells are the progeny of Dlx2 -expressing cells. By E16, PSA-NCAM-expressing cells also collect at the ventral aspect of the Zebrin II+ VZ wedge, the site of the nascent dorsolateral SVZ. A–C , Immunoreactivity for PSA-NCAM (TRITC) and Zebrin II (FITC) at the acute dorsolateral angle of the lateral ventricle. B, C, asterisk , The PSA-NCAM+ cell extends leading processes ventrodorsally. Dlx2/tauLacZ (β-gal) labels PSA-NCAM+ progeny of Dlx2-expressing cells at the ventral aspect of the VZ. D–H , Immunoreactivity for β-galactosidase (TRITC) and PSA-NCAM (FITC) within the LGE at E16 ( D–F ) and forming dorsolateral SVZ at P0 ( G–H ). H , Enlargement of boxed area in G . Scale bars: B , 50 μm; E, G , 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Subpallial Dlx2-Expressing Cells Give Rise to Astrocytes and Oligodendrocytes in the Cerebral Cortex and White Matter

    doi: 10.1523/JNEUROSCI.22-22-09821.2002

    Figure Lengend Snippet: PSA-NCAM+ cells are the progeny of Dlx2 -expressing cells. By E16, PSA-NCAM-expressing cells also collect at the ventral aspect of the Zebrin II+ VZ wedge, the site of the nascent dorsolateral SVZ. A–C , Immunoreactivity for PSA-NCAM (TRITC) and Zebrin II (FITC) at the acute dorsolateral angle of the lateral ventricle. B, C, asterisk , The PSA-NCAM+ cell extends leading processes ventrodorsally. Dlx2/tauLacZ (β-gal) labels PSA-NCAM+ progeny of Dlx2-expressing cells at the ventral aspect of the VZ. D–H , Immunoreactivity for β-galactosidase (TRITC) and PSA-NCAM (FITC) within the LGE at E16 ( D–F ) and forming dorsolateral SVZ at P0 ( G–H ). H , Enlargement of boxed area in G . Scale bars: B , 50 μm; E, G , 100 μm.

    Article Snippet: Immunohistochemistry with monoclonal antibodies against Zebrin II (1:100; gift from R. Hawkes, University of Calgary, Alberta, Canada), the polysialylated form of neural cell adhesion molecule (PSA-NCAM)/5A5 (1:5; gift from J. Dodd, Columbia University, New York, NY), RC2 (1:1; Developmental Studies Hybridoma Bank, Iowa City, IA), TUJ-1 (1:500; Babco, Richmond, CA), CNPase (1:200; Sigma), and polyclonal antibodies against Dlx2 (1:200; gift from J. Rubenstein, University of California, San Francisco, CA), astrocyte-specific glutamate transporter (GLAST) (1:1000; Chemicon, Temecula, CA), and β-galactosidase (β-gal) (1:1000; Cortex Biochemical, San Leandro, CA) was performed as follows.

    Techniques: Expressing

    Subpallium-derived cells defined by Dlx2/tauLacZ reporter expression adopt mature glial phenotypes in the postnatal dorsal forebrain (P6–P10). A–C , Immature β-gal+ cells (TRITC) positioned within the corpus callosum coexpress Zebrin II (FITC). D–F , β-gal+ cells (FITC) exhibiting a more differentiated astrocytic phenotype coexpress GFAP (TRITC) and associate with blood vessels (note asterisk in F ). β-gal+ cells coexpress CNPase and possess the morphology of mature oligodendroglia in the white matter ( G–I ) and cerebral cortex ( J–L ).

    Journal: The Journal of Neuroscience

    Article Title: Subpallial Dlx2-Expressing Cells Give Rise to Astrocytes and Oligodendrocytes in the Cerebral Cortex and White Matter

    doi: 10.1523/JNEUROSCI.22-22-09821.2002

    Figure Lengend Snippet: Subpallium-derived cells defined by Dlx2/tauLacZ reporter expression adopt mature glial phenotypes in the postnatal dorsal forebrain (P6–P10). A–C , Immature β-gal+ cells (TRITC) positioned within the corpus callosum coexpress Zebrin II (FITC). D–F , β-gal+ cells (FITC) exhibiting a more differentiated astrocytic phenotype coexpress GFAP (TRITC) and associate with blood vessels (note asterisk in F ). β-gal+ cells coexpress CNPase and possess the morphology of mature oligodendroglia in the white matter ( G–I ) and cerebral cortex ( J–L ).

    Article Snippet: Immunohistochemistry with monoclonal antibodies against Zebrin II (1:100; gift from R. Hawkes, University of Calgary, Alberta, Canada), the polysialylated form of neural cell adhesion molecule (PSA-NCAM)/5A5 (1:5; gift from J. Dodd, Columbia University, New York, NY), RC2 (1:1; Developmental Studies Hybridoma Bank, Iowa City, IA), TUJ-1 (1:500; Babco, Richmond, CA), CNPase (1:200; Sigma), and polyclonal antibodies against Dlx2 (1:200; gift from J. Rubenstein, University of California, San Francisco, CA), astrocyte-specific glutamate transporter (GLAST) (1:1000; Chemicon, Temecula, CA), and β-galactosidase (β-gal) (1:1000; Cortex Biochemical, San Leandro, CA) was performed as follows.

    Techniques: Derivative Assay, Expressing

    HCV core-induced TNF-α and IL-6 mediate the enhancement of HIV-1 infection in THP-1 macrophages and MDMs. (A) TNF-α and (B) IL-6 cytokine induction in naïve, HIV-BaL-infected, and/or stimulated with HCV core, and β-galactosidase protein control was measured by ELISA. (C) THP-1 macrophages and (D) MDMs were infected with HIV-BaL for 12 hours. Cells were left unstimulated or treated with HCV core, or β-galactosidase protein control, in the presence or absence of specific human monoclonal nAbs anti-TNF-α and/or anti-IL-6. HIV infectivity was determined by luciferase activity in cell lysates 48 hours after infection. Data are shown as relative fold induction of HIV infectivity (mean ± SD). Results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. *Significantly different ( P

    Journal: FEBS letters

    Article Title: Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK, and MEK1/2-dependent upregulation of TNF-α and IL-6

    doi: 10.1016/j.febslet.2014.08.009

    Figure Lengend Snippet: HCV core-induced TNF-α and IL-6 mediate the enhancement of HIV-1 infection in THP-1 macrophages and MDMs. (A) TNF-α and (B) IL-6 cytokine induction in naïve, HIV-BaL-infected, and/or stimulated with HCV core, and β-galactosidase protein control was measured by ELISA. (C) THP-1 macrophages and (D) MDMs were infected with HIV-BaL for 12 hours. Cells were left unstimulated or treated with HCV core, or β-galactosidase protein control, in the presence or absence of specific human monoclonal nAbs anti-TNF-α and/or anti-IL-6. HIV infectivity was determined by luciferase activity in cell lysates 48 hours after infection. Data are shown as relative fold induction of HIV infectivity (mean ± SD). Results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. *Significantly different ( P

    Article Snippet: THP-1 macrophages and MDMs were treated with either 5 μg/ml of recombinant HCV core protein genotype 1b fused to β-galactosidase (Virogen, Watertown, MA), or 5 μg/ml of β-Galactosidase (β-Gal) control protein (Virogen).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay

    HCV core enhances HIV-1 infectivity via TLR2. THP-1 macrophages (A) and MDMs (B) were infected with a luc-reporter, macrophage-tropic BaL HIV-1 pseudotype. Cells were unstimulated or stimulated with 5 μg/ml of HCV core or β-Gal recombinant protein control in the presence of a neutralizing human monoclonal anti-TLR2 Ab or isotype control (Invivogen) 12 hours after infection. Luc activity was measured 2 days after infection and results from 3 independent experiments (THP-1) and 2 independent MDMs donors are shown. Luciferase activity in cell lysates was measured as relative light units per second. Results represent means ± SD. To determine the role of MAP kinases in HCV core-mediated enhancement of HIV infection in THP-1 macrophages (C) and MDMs (D), cells were prestimulated with specific inhibitors of JNK (50 μM SP600125), p38 (20 μM SB203580), and MEK1/2 (50 μM PD98059, and 20 μM U0126) for 30 minutes and then infected with HIV-1 BaL pseudotype. Infected cells were left unstimulated or treated with HCV core or β-Gal 12 hours after infection. Luc activity was measured 2 days after infection and results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. Luciferase activity in cell lysates was measured as relative light units per second. Data are shown as relative luciferase units (RLU) of HIV infectivity (mean ± SD). Values that were significantly different ( P

    Journal: FEBS letters

    Article Title: Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK, and MEK1/2-dependent upregulation of TNF-α and IL-6

    doi: 10.1016/j.febslet.2014.08.009

    Figure Lengend Snippet: HCV core enhances HIV-1 infectivity via TLR2. THP-1 macrophages (A) and MDMs (B) were infected with a luc-reporter, macrophage-tropic BaL HIV-1 pseudotype. Cells were unstimulated or stimulated with 5 μg/ml of HCV core or β-Gal recombinant protein control in the presence of a neutralizing human monoclonal anti-TLR2 Ab or isotype control (Invivogen) 12 hours after infection. Luc activity was measured 2 days after infection and results from 3 independent experiments (THP-1) and 2 independent MDMs donors are shown. Luciferase activity in cell lysates was measured as relative light units per second. Results represent means ± SD. To determine the role of MAP kinases in HCV core-mediated enhancement of HIV infection in THP-1 macrophages (C) and MDMs (D), cells were prestimulated with specific inhibitors of JNK (50 μM SP600125), p38 (20 μM SB203580), and MEK1/2 (50 μM PD98059, and 20 μM U0126) for 30 minutes and then infected with HIV-1 BaL pseudotype. Infected cells were left unstimulated or treated with HCV core or β-Gal 12 hours after infection. Luc activity was measured 2 days after infection and results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. Luciferase activity in cell lysates was measured as relative light units per second. Data are shown as relative luciferase units (RLU) of HIV infectivity (mean ± SD). Values that were significantly different ( P

    Article Snippet: THP-1 macrophages and MDMs were treated with either 5 μg/ml of recombinant HCV core protein genotype 1b fused to β-galactosidase (Virogen, Watertown, MA), or 5 μg/ml of β-Galactosidase (β-Gal) control protein (Virogen).

    Techniques: Infection, Recombinant, Activity Assay, Luciferase

    HCV core enhances HIV-1 BaL infection of THP-1 macrophages and MDMs. (A) HCV core dose curve experiment. THP-1 differentiated cells were stimulated with 0.1, 0.5, 1.0, 2.5, and 5 μg/ml of HCV core, or β-Gal recombinant protein control at 12 hours after infection with a luc-reporter, macrophage-tropic BaL HIV-1 pseudotype. (B) HCV core time course experiment. THP-1 differentiated cells were stimulated with 5 μg/ml of HCV core, or β-Gal recombinant protein control at the time of infection (time 0) and 4, 8,12, and 24 hours after HIV-1 BaL infection. (C) Primary human macrophages (MDMs) were stimulated with 5 μg/ml of HCV core, or β-Gal recombinant protein control at 12 hours after HIV-1 BaL infection. LTR-driven Luciferase activity was measured 2 days after infection and results from 5 independent experiments (THP-1) and 2 independent MDMs donors are shown. Luciferase activity in cell lysates was measured as relative light units per second. Data are shown as percent luciferase expression relative to HIV-1 infected alone cells (mean ± SD). Values that were significantly different (P

    Journal: FEBS letters

    Article Title: Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK, and MEK1/2-dependent upregulation of TNF-α and IL-6

    doi: 10.1016/j.febslet.2014.08.009

    Figure Lengend Snippet: HCV core enhances HIV-1 BaL infection of THP-1 macrophages and MDMs. (A) HCV core dose curve experiment. THP-1 differentiated cells were stimulated with 0.1, 0.5, 1.0, 2.5, and 5 μg/ml of HCV core, or β-Gal recombinant protein control at 12 hours after infection with a luc-reporter, macrophage-tropic BaL HIV-1 pseudotype. (B) HCV core time course experiment. THP-1 differentiated cells were stimulated with 5 μg/ml of HCV core, or β-Gal recombinant protein control at the time of infection (time 0) and 4, 8,12, and 24 hours after HIV-1 BaL infection. (C) Primary human macrophages (MDMs) were stimulated with 5 μg/ml of HCV core, or β-Gal recombinant protein control at 12 hours after HIV-1 BaL infection. LTR-driven Luciferase activity was measured 2 days after infection and results from 5 independent experiments (THP-1) and 2 independent MDMs donors are shown. Luciferase activity in cell lysates was measured as relative light units per second. Data are shown as percent luciferase expression relative to HIV-1 infected alone cells (mean ± SD). Values that were significantly different (P

    Article Snippet: THP-1 macrophages and MDMs were treated with either 5 μg/ml of recombinant HCV core protein genotype 1b fused to β-galactosidase (Virogen, Watertown, MA), or 5 μg/ml of β-Galactosidase (β-Gal) control protein (Virogen).

    Techniques: Infection, Recombinant, Luciferase, Activity Assay, Expressing

    TNF-α and IL-6 cytokine induction mediated by HCV core is dependent on MAP kinases. THP-1 macrophages (A and B) and MDMs (C and D) were stimulated with HCV core or β-galactosidase protein control in the presence or absence of 50 μM SP600125 (JNK inhibitor), 20 μM SB203580 (p38 inhibitor), 50 μM PD98059 (MEK1/2 inhibitor), and 10 μM U0126 (MEK1/2 inhibitor). TNF-α (A and C) and IL-6 (B and D) induction was determined by ELISA (R D Systems, Minneapolis, MN) 14 hours after stimulation. Results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. Data are means ± SD. *Significantly different ( P

    Journal: FEBS letters

    Article Title: Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK, and MEK1/2-dependent upregulation of TNF-α and IL-6

    doi: 10.1016/j.febslet.2014.08.009

    Figure Lengend Snippet: TNF-α and IL-6 cytokine induction mediated by HCV core is dependent on MAP kinases. THP-1 macrophages (A and B) and MDMs (C and D) were stimulated with HCV core or β-galactosidase protein control in the presence or absence of 50 μM SP600125 (JNK inhibitor), 20 μM SB203580 (p38 inhibitor), 50 μM PD98059 (MEK1/2 inhibitor), and 10 μM U0126 (MEK1/2 inhibitor). TNF-α (A and C) and IL-6 (B and D) induction was determined by ELISA (R D Systems, Minneapolis, MN) 14 hours after stimulation. Results from 3 independent experiments (THP-1) and 2 independent MDM donors are shown. Data are means ± SD. *Significantly different ( P

    Article Snippet: THP-1 macrophages and MDMs were treated with either 5 μg/ml of recombinant HCV core protein genotype 1b fused to β-galactosidase (Virogen, Watertown, MA), or 5 μg/ml of β-Galactosidase (β-Gal) control protein (Virogen).

    Techniques: Enzyme-linked Immunosorbent Assay