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  • 99
    ATCC p aeruginosa atcc 27853
    P Aeruginosa Atcc 27853, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 4021 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pvdf membranes
    Pvdf Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 56664 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nonidet p 40
    Nonidet P 40, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14485 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore immobilon p polyvinylidene difluoride membranes
    Immobilon P Polyvinylidene Difluoride Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore immobilon p
    Immobilon P, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 5384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore p nitrophenyl phosphate
    P Nitrophenyl Phosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6461 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Marinus p marinus
    P Marinus, supplied by Marinus, used in various techniques. Bioz Stars score: 92/100, based on 2900 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p akt
    P Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 18276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Umetrics simca p
    PCA models of the four biological batches ( a R 2 X = 0.73, Q 2 X = 0.63, b R 2 X = 0.78, Q 2 X = 0.58, c R 2 X = 0.78, Q 2 X = 0.53, d R 2 X = 0.84, Q 2 X = 0.64) displaying untreated control cell ( open circles ) samples, high (1000 µM) BMAA concentration ( filled inverted triangles ) samples, medium (500 µM) BMAA concentration ( filled squares ) samples and low (50 µM) BMAA concentration ( filled upright triangles ) samples obtained from LC–MS analysis in positive ionization mode. Each sample was injected three times in a random order throughout the analytical run. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the <t>SIMCA-P+</t> software. Moreover, only features with CV values below 30% were included in the analysis
    Simca P, supplied by Umetrics, used in various techniques. Bioz Stars score: 92/100, based on 2570 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p mtor
    PCA models of the four biological batches ( a R 2 X = 0.73, Q 2 X = 0.63, b R 2 X = 0.78, Q 2 X = 0.58, c R 2 X = 0.78, Q 2 X = 0.53, d R 2 X = 0.84, Q 2 X = 0.64) displaying untreated control cell ( open circles ) samples, high (1000 µM) BMAA concentration ( filled inverted triangles ) samples, medium (500 µM) BMAA concentration ( filled squares ) samples and low (50 µM) BMAA concentration ( filled upright triangles ) samples obtained from LC–MS analysis in positive ionization mode. Each sample was injected three times in a random order throughout the analytical run. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the <t>SIMCA-P+</t> software. Moreover, only features with CV values below 30% were included in the analysis
    P Mtor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC p gingivalis atcc 33277
    Macrophage migration-inhibitory factor (MIF) acted as a regulator in Porphyromonas <t>gingivalis</t> ( P. gingivalis )-induced intercellular adhesion molecule-1 (ICAM-1) expression. a Western blot analysis of ICAM-1 in EA.hy926 cells. b Quantitative analyses of the optical density relative to the internal reference protein GAPDH in Western blot analysis. c Quantitative Real-time PCR analysis of ICAM-1 mRNA. Infection with P. gingivalis <t>ATCC</t> 33277 (MOI = 100, 24 h) could induce the expression of ICAM-1 and ICAM-1 mRNA in EA.hy926 cells, this inductive effect of P. gingivalis was blocked by the MIF antagonist ISO-1. Sufficient exogenous recombinant MIF (rMIF) supplementation rescued the ICAM-1 expression. Data are presented as means ± SD from three independent experiments. * P
    P Gingivalis Atcc 33277, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC p aeruginosa
    Caspase-1 was essential for disease progression and bacterial clearance in Pseudomonas <t>aeruginosa</t> keratitis. (A–F) C57BL/6 mice were subconjunctivally injected with caspase-1 siRNA or scrambled control, and then infected with PA routinely. (G–J) C57BL/6 mice were subconjunctivally injected with the caspase-1 inhibitor Ac-YVAD-CMK (YVAD) or vehicle control (DMSO), and then infected with P. aeruginosa routinely. (A) mRNA levels of caspase-1 was examined by real-time PCR in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (B) Protein levels of caspase-1 were detected with western blot in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (C,G) Clinical score was recorded for each cornea at 1, 3, and 5 days after infection. Representative photographs of infected eyes in caspase-1 siRNA- (E) versus control- (D) , YVAD (I) versus DMSO (H) treated mice were taken at 5 days after infection. (F,J) Bacterial load in the infected corneas was examined by plate count assay at 1 and 5 days after infection. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (C–E,G–I) or five mice per group (A,B,F,J) . * P
    P Aeruginosa, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 4617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p stat3
    Caspase-1 was essential for disease progression and bacterial clearance in Pseudomonas <t>aeruginosa</t> keratitis. (A–F) C57BL/6 mice were subconjunctivally injected with caspase-1 siRNA or scrambled control, and then infected with PA routinely. (G–J) C57BL/6 mice were subconjunctivally injected with the caspase-1 inhibitor Ac-YVAD-CMK (YVAD) or vehicle control (DMSO), and then infected with P. aeruginosa routinely. (A) mRNA levels of caspase-1 was examined by real-time PCR in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (B) Protein levels of caspase-1 were detected with western blot in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (C,G) Clinical score was recorded for each cornea at 1, 3, and 5 days after infection. Representative photographs of infected eyes in caspase-1 siRNA- (E) versus control- (D) , YVAD (I) versus DMSO (H) treated mice were taken at 5 days after infection. (F,J) Bacterial load in the infected corneas was examined by plate count assay at 1 and 5 days after infection. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (C–E,G–I) or five mice per group (A,B,F,J) . * P
    P Stat3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5675 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore taxol
    Caspase-1 was essential for disease progression and bacterial clearance in Pseudomonas <t>aeruginosa</t> keratitis. (A–F) C57BL/6 mice were subconjunctivally injected with caspase-1 siRNA or scrambled control, and then infected with PA routinely. (G–J) C57BL/6 mice were subconjunctivally injected with the caspase-1 inhibitor Ac-YVAD-CMK (YVAD) or vehicle control (DMSO), and then infected with P. aeruginosa routinely. (A) mRNA levels of caspase-1 was examined by real-time PCR in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (B) Protein levels of caspase-1 were detected with western blot in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (C,G) Clinical score was recorded for each cornea at 1, 3, and 5 days after infection. Representative photographs of infected eyes in caspase-1 siRNA- (E) versus control- (D) , YVAD (I) versus DMSO (H) treated mice were taken at 5 days after infection. (F,J) Bacterial load in the infected corneas was examined by plate count assay at 1 and 5 days after infection. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (C–E,G–I) or five mice per group (A,B,F,J) . * P
    Taxol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher penicillin streptomycin p s
    Caspase-1 was essential for disease progression and bacterial clearance in Pseudomonas <t>aeruginosa</t> keratitis. (A–F) C57BL/6 mice were subconjunctivally injected with caspase-1 siRNA or scrambled control, and then infected with PA routinely. (G–J) C57BL/6 mice were subconjunctivally injected with the caspase-1 inhibitor Ac-YVAD-CMK (YVAD) or vehicle control (DMSO), and then infected with P. aeruginosa routinely. (A) mRNA levels of caspase-1 was examined by real-time PCR in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (B) Protein levels of caspase-1 were detected with western blot in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (C,G) Clinical score was recorded for each cornea at 1, 3, and 5 days after infection. Representative photographs of infected eyes in caspase-1 siRNA- (E) versus control- (D) , YVAD (I) versus DMSO (H) treated mice were taken at 5 days after infection. (F,J) Bacterial load in the infected corneas was examined by plate count assay at 1 and 5 days after infection. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (C–E,G–I) or five mice per group (A,B,F,J) . * P
    Penicillin Streptomycin P S, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1809 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Umetrics simca p software
    OPLS-DA analysis of TTGE profiles, according to disease severity. Panel A) On the left are reported representative TTGE profiles from severe and mild CF patients. <t>Simca-P+</t> software was used to compute weight coefficients for each TTGE band (87 x variables) on the two subgroups of CF patients (2 y variables) harbouring at least one F508del mutation in one allele: severe (S) and mild (M), with a scaled a centred data set. A clustered image heatmap was generated with CIMminer online software (panel A, right). As depicted in the color-coded legend, the higher that the coefficient value is, the higher the weight (red), while the lower that the value is, the lower the weight (turquoise). Panel B) Faecal TTGE profiles of all 24 CF patients harbouring at least one F508del allele were analysed by OPLS-DA, and the resulting 3D score plot model gave a significant separation among the two sub-groups: severe (S, black diamonds) and mild (M, red diamonds).
    Simca P Software, supplied by Umetrics, used in various techniques. Bioz Stars score: 92/100, based on 1843 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore p coumaric acid
    OPLS-DA analysis of TTGE profiles, according to disease severity. Panel A) On the left are reported representative TTGE profiles from severe and mild CF patients. <t>Simca-P+</t> software was used to compute weight coefficients for each TTGE band (87 x variables) on the two subgroups of CF patients (2 y variables) harbouring at least one F508del mutation in one allele: severe (S) and mild (M), with a scaled a centred data set. A clustered image heatmap was generated with CIMminer online software (panel A, right). As depicted in the color-coded legend, the higher that the coefficient value is, the higher the weight (red), while the lower that the value is, the lower the weight (turquoise). Panel B) Faecal TTGE profiles of all 24 CF patients harbouring at least one F508del allele were analysed by OPLS-DA, and the resulting 3D score plot model gave a significant separation among the two sub-groups: severe (S, black diamonds) and mild (M, red diamonds).
    P Coumaric Acid, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer α 32 p dctp
    The influence of exonuclease activity on rNMP incorporation by Pol γ. ( A ) Schematic view of reaction set up in Fig 3B. ( B ) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo - ) Pol γ. In vitro replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α- 32 <t>P]-dCTP.</t> Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. ( C ) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo - (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. ( D ) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT PolgA (n = 2) and exonuclease-deficient PolgA D257A (n = 3; “exo - ”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). ( E ) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT PolgA and PolgA D257A mice.
    α 32 P Dctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 2679 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bellen Chemistry verstreken p
    The influence of exonuclease activity on rNMP incorporation by Pol γ. ( A ) Schematic view of reaction set up in Fig 3B. ( B ) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo - ) Pol γ. In vitro replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α- 32 <t>P]-dCTP.</t> Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. ( C ) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo - (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. ( D ) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT PolgA (n = 2) and exonuclease-deficient PolgA D257A (n = 3; “exo - ”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). ( E ) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT PolgA and PolgA D257A mice.
    Verstreken P, supplied by Bellen Chemistry, used in various techniques. Bioz Stars score: 92/100, based on 658 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p ampk
    The influence of exonuclease activity on rNMP incorporation by Pol γ. ( A ) Schematic view of reaction set up in Fig 3B. ( B ) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo - ) Pol γ. In vitro replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α- 32 <t>P]-dCTP.</t> Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. ( C ) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo - (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. ( D ) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT PolgA (n = 2) and exonuclease-deficient PolgA D257A (n = 3; “exo - ”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). ( E ) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT PolgA and PolgA D257A mice.
    P Ampk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2534 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    JASCO Inc jasco p 2000 polarimeter
    The influence of exonuclease activity on rNMP incorporation by Pol γ. ( A ) Schematic view of reaction set up in Fig 3B. ( B ) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo - ) Pol γ. In vitro replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α- 32 <t>P]-dCTP.</t> Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. ( C ) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo - (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. ( D ) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT PolgA (n = 2) and exonuclease-deficient PolgA D257A (n = 3; “exo - ”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). ( E ) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT PolgA and PolgA D257A mice.
    Jasco P 2000 Polarimeter, supplied by JASCO Inc, used in various techniques. Bioz Stars score: 96/100, based on 1154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare hybond p membranes
    The influence of exonuclease activity on rNMP incorporation by Pol γ. ( A ) Schematic view of reaction set up in Fig 3B. ( B ) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo - ) Pol γ. In vitro replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α- 32 <t>P]-dCTP.</t> Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. ( C ) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo - (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. ( D ) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT PolgA (n = 2) and exonuclease-deficient PolgA D257A (n = 3; “exo - ”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). ( E ) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT PolgA and PolgA D257A mice.
    Hybond P Membranes, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1929 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p iκbα
    The anti-inflammatory and anti-fibrotic effects of 5-MTP in vitro. a , b The protein expression and relative quantitative data of <t>IκBα,</t> p-IκBα, NF-κB p65, COX-2 and MCP-1 as well as Nrf2, HO-1 and NQO-1 in HK-2 cells induced by TGF-β1 as indicated. * P
    P Iκbα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    The P 2000 WI system includes a tungsten halogen lamp and 589 nm inference filter for routine pharmaceutical measurements with the flexibility to add additional filters as applications and needs
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    Image Search Results


    PCA models of the four biological batches ( a R 2 X = 0.73, Q 2 X = 0.63, b R 2 X = 0.78, Q 2 X = 0.58, c R 2 X = 0.78, Q 2 X = 0.53, d R 2 X = 0.84, Q 2 X = 0.64) displaying untreated control cell ( open circles ) samples, high (1000 µM) BMAA concentration ( filled inverted triangles ) samples, medium (500 µM) BMAA concentration ( filled squares ) samples and low (50 µM) BMAA concentration ( filled upright triangles ) samples obtained from LC–MS analysis in positive ionization mode. Each sample was injected three times in a random order throughout the analytical run. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the SIMCA-P+ software. Moreover, only features with CV values below 30% were included in the analysis

    Journal: Amino Acids

    Article Title: β-N-Methylamino-l-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling

    doi: 10.1007/s00726-017-2391-8

    Figure Lengend Snippet: PCA models of the four biological batches ( a R 2 X = 0.73, Q 2 X = 0.63, b R 2 X = 0.78, Q 2 X = 0.58, c R 2 X = 0.78, Q 2 X = 0.53, d R 2 X = 0.84, Q 2 X = 0.64) displaying untreated control cell ( open circles ) samples, high (1000 µM) BMAA concentration ( filled inverted triangles ) samples, medium (500 µM) BMAA concentration ( filled squares ) samples and low (50 µM) BMAA concentration ( filled upright triangles ) samples obtained from LC–MS analysis in positive ionization mode. Each sample was injected three times in a random order throughout the analytical run. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the SIMCA-P+ software. Moreover, only features with CV values below 30% were included in the analysis

    Article Snippet: Univariate and multivariate data analysis Positive and negative LC–MS data as well as NMR data were normalized to total intensity in Microsoft Excel and separately analyzed by multivariate data analysis using the SIMCA-P+ (version 13.0, Umetrics, Umeå, Sweden) computational software package.

    Techniques: Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Injection, Software

    PCA model ( R 2 X = 0.79, Q 2 X = 0.63) displaying untreated control cells ( open circles ) vs BMAA exposed cells (low (50 µM), medium (250 µM) and high (1000 µM) concentration, filled circles ) obtained from LC–MS analysis in positive mode. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the SIMCA-P+ software. Moreover, only features with CV values below 30% were included in the analysis

    Journal: Amino Acids

    Article Title: β-N-Methylamino-l-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling

    doi: 10.1007/s00726-017-2391-8

    Figure Lengend Snippet: PCA model ( R 2 X = 0.79, Q 2 X = 0.63) displaying untreated control cells ( open circles ) vs BMAA exposed cells (low (50 µM), medium (250 µM) and high (1000 µM) concentration, filled circles ) obtained from LC–MS analysis in positive mode. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the SIMCA-P+ software. Moreover, only features with CV values below 30% were included in the analysis

    Article Snippet: Univariate and multivariate data analysis Positive and negative LC–MS data as well as NMR data were normalized to total intensity in Microsoft Excel and separately analyzed by multivariate data analysis using the SIMCA-P+ (version 13.0, Umetrics, Umeå, Sweden) computational software package.

    Techniques: Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Software

    Macrophage migration-inhibitory factor (MIF) acted as a regulator in Porphyromonas gingivalis ( P. gingivalis )-induced intercellular adhesion molecule-1 (ICAM-1) expression. a Western blot analysis of ICAM-1 in EA.hy926 cells. b Quantitative analyses of the optical density relative to the internal reference protein GAPDH in Western blot analysis. c Quantitative Real-time PCR analysis of ICAM-1 mRNA. Infection with P. gingivalis ATCC 33277 (MOI = 100, 24 h) could induce the expression of ICAM-1 and ICAM-1 mRNA in EA.hy926 cells, this inductive effect of P. gingivalis was blocked by the MIF antagonist ISO-1. Sufficient exogenous recombinant MIF (rMIF) supplementation rescued the ICAM-1 expression. Data are presented as means ± SD from three independent experiments. * P

    Journal: BMC Microbiology

    Article Title: Porphyromonas gingivalis ATCC 33277 promotes intercellular adhesion molecule-1 expression in endothelial cells and monocyte-endothelial cell adhesion through macrophage migration inhibitory factor

    doi: 10.1186/s12866-018-1156-1

    Figure Lengend Snippet: Macrophage migration-inhibitory factor (MIF) acted as a regulator in Porphyromonas gingivalis ( P. gingivalis )-induced intercellular adhesion molecule-1 (ICAM-1) expression. a Western blot analysis of ICAM-1 in EA.hy926 cells. b Quantitative analyses of the optical density relative to the internal reference protein GAPDH in Western blot analysis. c Quantitative Real-time PCR analysis of ICAM-1 mRNA. Infection with P. gingivalis ATCC 33277 (MOI = 100, 24 h) could induce the expression of ICAM-1 and ICAM-1 mRNA in EA.hy926 cells, this inductive effect of P. gingivalis was blocked by the MIF antagonist ISO-1. Sufficient exogenous recombinant MIF (rMIF) supplementation rescued the ICAM-1 expression. Data are presented as means ± SD from three independent experiments. * P

    Article Snippet: In contrast, cell adhesion was decreased in ISO-1-treated cells compared with those infected with P. gingivalis ATCC 33277 ( P < 0.01).

    Techniques: Migration, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Infection, Recombinant

    Macrophage migration-inhibitory factor (MIF) regulated the increased monocyte adhesion to endothelial cells infected with Porphyromonas gingivalis ( P. gingivalis ) . a THP-1 cell adhesion to EA.hy926 cells was labeled by calcein-AM and visualized. Pictures are representative fields captured by flurencence microscope (upper line) or microscope (lower line) of three independent experiments (magnification × 100). b The histogram of the evaluation of adhered THP-1 cell assessed by cell count assay. Compared with uninfected cells, P. gingivalis ATCC 33277 infection (MOI = 100, 24 h) markedly increased THP-1 cell adhesion to endothelial cells ( P

    Journal: BMC Microbiology

    Article Title: Porphyromonas gingivalis ATCC 33277 promotes intercellular adhesion molecule-1 expression in endothelial cells and monocyte-endothelial cell adhesion through macrophage migration inhibitory factor

    doi: 10.1186/s12866-018-1156-1

    Figure Lengend Snippet: Macrophage migration-inhibitory factor (MIF) regulated the increased monocyte adhesion to endothelial cells infected with Porphyromonas gingivalis ( P. gingivalis ) . a THP-1 cell adhesion to EA.hy926 cells was labeled by calcein-AM and visualized. Pictures are representative fields captured by flurencence microscope (upper line) or microscope (lower line) of three independent experiments (magnification × 100). b The histogram of the evaluation of adhered THP-1 cell assessed by cell count assay. Compared with uninfected cells, P. gingivalis ATCC 33277 infection (MOI = 100, 24 h) markedly increased THP-1 cell adhesion to endothelial cells ( P

    Article Snippet: In contrast, cell adhesion was decreased in ISO-1-treated cells compared with those infected with P. gingivalis ATCC 33277 ( P < 0.01).

    Techniques: Migration, Infection, Labeling, Microscopy, Cell Counting

    Porphyromonas gingivalis ( P. gingivalis ) ATCC 33277 infection enhances MIF secretion in EA.hy926 cells . EA.hy926 were challenged with Escherichia coli ( E. coli ) lipopolysaccharide (LPS, 1 μg/mL) or P. gingivalis (MOI = 100) for 4, 10 or 24 h. MIF levels was analyzed using ELISA. Infection of P. gingivalis significantly increased MIF secretion in EA.hy926 cells. EA.hy926 were incubated with medium only as a control. E.coli LPS was used as a positive control. The I bar shows the standard deviation. * P

    Journal: BMC Microbiology

    Article Title: Porphyromonas gingivalis ATCC 33277 promotes intercellular adhesion molecule-1 expression in endothelial cells and monocyte-endothelial cell adhesion through macrophage migration inhibitory factor

    doi: 10.1186/s12866-018-1156-1

    Figure Lengend Snippet: Porphyromonas gingivalis ( P. gingivalis ) ATCC 33277 infection enhances MIF secretion in EA.hy926 cells . EA.hy926 were challenged with Escherichia coli ( E. coli ) lipopolysaccharide (LPS, 1 μg/mL) or P. gingivalis (MOI = 100) for 4, 10 or 24 h. MIF levels was analyzed using ELISA. Infection of P. gingivalis significantly increased MIF secretion in EA.hy926 cells. EA.hy926 were incubated with medium only as a control. E.coli LPS was used as a positive control. The I bar shows the standard deviation. * P

    Article Snippet: In contrast, cell adhesion was decreased in ISO-1-treated cells compared with those infected with P. gingivalis ATCC 33277 ( P < 0.01).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Standard Deviation

    Caspase-1 was essential for disease progression and bacterial clearance in Pseudomonas aeruginosa keratitis. (A–F) C57BL/6 mice were subconjunctivally injected with caspase-1 siRNA or scrambled control, and then infected with PA routinely. (G–J) C57BL/6 mice were subconjunctivally injected with the caspase-1 inhibitor Ac-YVAD-CMK (YVAD) or vehicle control (DMSO), and then infected with P. aeruginosa routinely. (A) mRNA levels of caspase-1 was examined by real-time PCR in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (B) Protein levels of caspase-1 were detected with western blot in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (C,G) Clinical score was recorded for each cornea at 1, 3, and 5 days after infection. Representative photographs of infected eyes in caspase-1 siRNA- (E) versus control- (D) , YVAD (I) versus DMSO (H) treated mice were taken at 5 days after infection. (F,J) Bacterial load in the infected corneas was examined by plate count assay at 1 and 5 days after infection. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (C–E,G–I) or five mice per group (A,B,F,J) . * P

    Journal: Frontiers in Immunology

    Article Title: Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis

    doi: 10.3389/fimmu.2018.01121

    Figure Lengend Snippet: Caspase-1 was essential for disease progression and bacterial clearance in Pseudomonas aeruginosa keratitis. (A–F) C57BL/6 mice were subconjunctivally injected with caspase-1 siRNA or scrambled control, and then infected with PA routinely. (G–J) C57BL/6 mice were subconjunctivally injected with the caspase-1 inhibitor Ac-YVAD-CMK (YVAD) or vehicle control (DMSO), and then infected with P. aeruginosa routinely. (A) mRNA levels of caspase-1 was examined by real-time PCR in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (B) Protein levels of caspase-1 were detected with western blot in caspase-1 siRNA- versus control-treated corneas at 1 and 5 days after infection. (C,G) Clinical score was recorded for each cornea at 1, 3, and 5 days after infection. Representative photographs of infected eyes in caspase-1 siRNA- (E) versus control- (D) , YVAD (I) versus DMSO (H) treated mice were taken at 5 days after infection. (F,J) Bacterial load in the infected corneas was examined by plate count assay at 1 and 5 days after infection. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (C–E,G–I) or five mice per group (A,B,F,J) . * P

    Article Snippet: P. aeruginosa (ATCC 19660) was killed by heating 100 μl aliquots of bacteria solution [108 colony forming units (CFU)/ml] at 65°C for an hour.

    Techniques: Mouse Assay, Injection, Infection, Real-time Polymerase Chain Reaction, Western Blot

    Triggering receptors expressed on myeloid cells 2 (TREM-2) inhibited caspase-1-dependent pyroptosis by mediating the NLRP3 inflammasome. (A) Immunoblot analysis of cleaved caspase-1 (p20) in culture supernatants of LPS-primed wild type (WT) and Trem2 −/− bone marrow-derived macrophages (BMDMs) stimulated with nigericin, immunoblot analysis of the precursors of caspase-1, and NLRP3 in lysates of those cells (Input). (B) IL-1β in supernatants from LPS-primed WT and Trem2 −/− BMDMs stimulated with nigericin were tested by enzyme-linked immunosorbent assay. (C) BMDMs from WT C57BL/6 mice were treated with heat-killed P. aeruginosa at MOI of 5 for 6 h or stimulated with nigericin, followed by immunoprecipitation using anti-TREM2 antibody. The immunoprecipitates were further immunoblotted with anti-TREM2, anti-caspase-1, and anti-NLRP3 antibodies. The blot shows the detection of caspase-1 and NLRP3 in the TREM2 immunoprecipitates. (D) Normal and infected corneas from WT C57BL/6 mice were harvested at 5 days after Pseudomonas aeruginosa infection, followed by immunoprecipitation using anti-TREM2 antibody. The immunoprecipitates were further immunoblotted with anti-TREM2, anti-caspase-1, and anti-NLRP3 antibodies. The blot shows the detection of caspase-1 and NLRP3 in the TREM2 immunoprecipitates. Data were the mean ± SEM and represent three individual experiments. * P

    Journal: Frontiers in Immunology

    Article Title: Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis

    doi: 10.3389/fimmu.2018.01121

    Figure Lengend Snippet: Triggering receptors expressed on myeloid cells 2 (TREM-2) inhibited caspase-1-dependent pyroptosis by mediating the NLRP3 inflammasome. (A) Immunoblot analysis of cleaved caspase-1 (p20) in culture supernatants of LPS-primed wild type (WT) and Trem2 −/− bone marrow-derived macrophages (BMDMs) stimulated with nigericin, immunoblot analysis of the precursors of caspase-1, and NLRP3 in lysates of those cells (Input). (B) IL-1β in supernatants from LPS-primed WT and Trem2 −/− BMDMs stimulated with nigericin were tested by enzyme-linked immunosorbent assay. (C) BMDMs from WT C57BL/6 mice were treated with heat-killed P. aeruginosa at MOI of 5 for 6 h or stimulated with nigericin, followed by immunoprecipitation using anti-TREM2 antibody. The immunoprecipitates were further immunoblotted with anti-TREM2, anti-caspase-1, and anti-NLRP3 antibodies. The blot shows the detection of caspase-1 and NLRP3 in the TREM2 immunoprecipitates. (D) Normal and infected corneas from WT C57BL/6 mice were harvested at 5 days after Pseudomonas aeruginosa infection, followed by immunoprecipitation using anti-TREM2 antibody. The immunoprecipitates were further immunoblotted with anti-TREM2, anti-caspase-1, and anti-NLRP3 antibodies. The blot shows the detection of caspase-1 and NLRP3 in the TREM2 immunoprecipitates. Data were the mean ± SEM and represent three individual experiments. * P

    Article Snippet: P. aeruginosa (ATCC 19660) was killed by heating 100 μl aliquots of bacteria solution [108 colony forming units (CFU)/ml] at 65°C for an hour.

    Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Immunoprecipitation, Infection

    Triggering receptors expressed on myeloid cells 2 (TREM-2) promoted host resistance against Pseudomonas aeruginosa keratitis via inhibiting caspase-1-dependent pyroptosis. Wild type (WT) mice were subconjunctivally injected with the vehicle control (DMSO) and Trem2 −/− mice were subconjunctivally injected with the vehicle control (DMSO) or caspase-1 inhibitor Ac-YVAD-CMK (YVAD), and then infected with P. aeruginosa routinely. mRNA expression levels of cytokines, including IL-1β (A) , IL-18 (C) , TNF-α (D) were examined by real-time PCR in the infected corneas at 1 and 5 days after infection. (B) Protein level of IL-1β in infected corneas was tested by enzyme-linked immunosorbent assay at 1 and 5 days after infection. (E) WT and Trem2 −/− BMDM were treated with YVAD (40 µM) or DMSO for 1 h, followed by heat-killed P. aeruginosa treatment at MOI of 5 for 6 h. mRNA expression levels of IL-1β and IL-18 were examined by real-time PCR. (F) The protein levels of GSDMD and its N-terminal domain in corneas were detected by western blot. Data were the mean ± SEM and represent three individual experiments each with five mice per group. * P

    Journal: Frontiers in Immunology

    Article Title: Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis

    doi: 10.3389/fimmu.2018.01121

    Figure Lengend Snippet: Triggering receptors expressed on myeloid cells 2 (TREM-2) promoted host resistance against Pseudomonas aeruginosa keratitis via inhibiting caspase-1-dependent pyroptosis. Wild type (WT) mice were subconjunctivally injected with the vehicle control (DMSO) and Trem2 −/− mice were subconjunctivally injected with the vehicle control (DMSO) or caspase-1 inhibitor Ac-YVAD-CMK (YVAD), and then infected with P. aeruginosa routinely. mRNA expression levels of cytokines, including IL-1β (A) , IL-18 (C) , TNF-α (D) were examined by real-time PCR in the infected corneas at 1 and 5 days after infection. (B) Protein level of IL-1β in infected corneas was tested by enzyme-linked immunosorbent assay at 1 and 5 days after infection. (E) WT and Trem2 −/− BMDM were treated with YVAD (40 µM) or DMSO for 1 h, followed by heat-killed P. aeruginosa treatment at MOI of 5 for 6 h. mRNA expression levels of IL-1β and IL-18 were examined by real-time PCR. (F) The protein levels of GSDMD and its N-terminal domain in corneas were detected by western blot. Data were the mean ± SEM and represent three individual experiments each with five mice per group. * P

    Article Snippet: P. aeruginosa (ATCC 19660) was killed by heating 100 μl aliquots of bacteria solution [108 colony forming units (CFU)/ml] at 65°C for an hour.

    Techniques: Mouse Assay, Injection, Infection, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

    Triggering receptors expressed on myeloid cells 2 (TREM-2) inhibited pro-inflammatory cytokine expression after Pseudomonas aeruginosa infection. mRNA expression levels of pro-inflammatory cytokines, including IL-1β (A) , IL-18 (C) , tumor necrosis factor-alpha (D) , IFN-γ (E) , and MIP-2 (F) was examined by real-time PCR in infected wild type (WT) and Trem2 −/− B6 corneas at 1 and 5 days after infection. (B) Protein level of IL-1β in WT and Trem2 −/− -infected B6 corneas was tested by enzyme-linked immunosorbent assay at 1 and 5 days after infection. mRNA expression levels of IL-1β, IL-18, and MIP-2 was examined by real-time PCR in heat-killed P. aeruginos a (HK-PA)-treated WT and Trem2 −/− B6 corneas at 5 days after treatment (G) and WT and Trem2 −/− BMDM (H) which were treatment with HK-PA at an MOI of 5 for 6 h. Data are the mean ± SEM and represent three individual experiments each with five mice per group. * P

    Journal: Frontiers in Immunology

    Article Title: Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis

    doi: 10.3389/fimmu.2018.01121

    Figure Lengend Snippet: Triggering receptors expressed on myeloid cells 2 (TREM-2) inhibited pro-inflammatory cytokine expression after Pseudomonas aeruginosa infection. mRNA expression levels of pro-inflammatory cytokines, including IL-1β (A) , IL-18 (C) , tumor necrosis factor-alpha (D) , IFN-γ (E) , and MIP-2 (F) was examined by real-time PCR in infected wild type (WT) and Trem2 −/− B6 corneas at 1 and 5 days after infection. (B) Protein level of IL-1β in WT and Trem2 −/− -infected B6 corneas was tested by enzyme-linked immunosorbent assay at 1 and 5 days after infection. mRNA expression levels of IL-1β, IL-18, and MIP-2 was examined by real-time PCR in heat-killed P. aeruginos a (HK-PA)-treated WT and Trem2 −/− B6 corneas at 5 days after treatment (G) and WT and Trem2 −/− BMDM (H) which were treatment with HK-PA at an MOI of 5 for 6 h. Data are the mean ± SEM and represent three individual experiments each with five mice per group. * P

    Article Snippet: P. aeruginosa (ATCC 19660) was killed by heating 100 μl aliquots of bacteria solution [108 colony forming units (CFU)/ml] at 65°C for an hour.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Mouse Assay

    Triggering receptors expressed on myeloid cells 2 (TREM-2) inhibited the expression and activation of caspase-1 after Pseudomonas aeruginosa infection. mRNA expression level of caspase-1 (A) was examined by real-time PCR in infected wild type (WT) and triggering receptors expressed on myeloid cells 2 ( Trem2 ) −/− B6 corneas at 1 and 5 days after infection. (B) The protein levels of total and cleaved caspase-1, NLRP3, and ASC in infected WT and Trem2 −/− B6 corneas were detected with western blot at 1 and 5 days after infection. (C) Caspase-1 protein expression was also determined by using immune-histochemistry in WT (left) and Trem2 −/− (right) B6 corneas after P. aeruginosa infection. Magnification was 100× at top panel and 200× at bottom panel, respectively. (D) FAM-FLICA staining was used to detect activated caspase-1 in macrophages (left), polymorphonuclear neutrophils (middle), and dendritic cells (right) from the infected WT and Trem2 −/− corneas at 5 days postinfection. (E) The mean fluorescence intensity for activated caspase-1 was analyzed using FlowJo software. Data were the mean ± SEM and represent three individual experiments each with five mice per group. * P

    Journal: Frontiers in Immunology

    Article Title: Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis

    doi: 10.3389/fimmu.2018.01121

    Figure Lengend Snippet: Triggering receptors expressed on myeloid cells 2 (TREM-2) inhibited the expression and activation of caspase-1 after Pseudomonas aeruginosa infection. mRNA expression level of caspase-1 (A) was examined by real-time PCR in infected wild type (WT) and triggering receptors expressed on myeloid cells 2 ( Trem2 ) −/− B6 corneas at 1 and 5 days after infection. (B) The protein levels of total and cleaved caspase-1, NLRP3, and ASC in infected WT and Trem2 −/− B6 corneas were detected with western blot at 1 and 5 days after infection. (C) Caspase-1 protein expression was also determined by using immune-histochemistry in WT (left) and Trem2 −/− (right) B6 corneas after P. aeruginosa infection. Magnification was 100× at top panel and 200× at bottom panel, respectively. (D) FAM-FLICA staining was used to detect activated caspase-1 in macrophages (left), polymorphonuclear neutrophils (middle), and dendritic cells (right) from the infected WT and Trem2 −/− corneas at 5 days postinfection. (E) The mean fluorescence intensity for activated caspase-1 was analyzed using FlowJo software. Data were the mean ± SEM and represent three individual experiments each with five mice per group. * P

    Article Snippet: P. aeruginosa (ATCC 19660) was killed by heating 100 μl aliquots of bacteria solution [108 colony forming units (CFU)/ml] at 65°C for an hour.

    Techniques: Expressing, Activation Assay, Infection, Real-time Polymerase Chain Reaction, Western Blot, Staining, Fluorescence, Software, Mouse Assay

    Triggering receptors expressed on myeloid cells 2 (TREM-2) inhibited pyroptosis of infiltrating cells in Pseudomonas aeruginosa keratitis. Pyroptosis in the infected cornea assessed with terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining. TUNEL-positive staining (green) was detected in Trem2 −/− (A) versus wild type (WT) (B) corneas at 5 days after infection. Cell nuclei were stained with 4,6-diamino-2-phenyl indole (DAPI; blue). Magnification was 200 and 400× respectively. (C) The protein levels of GSDMD and its N-terminal domain in Trem2 −/− versus WT B6 corneas were detected with western blot. (D) The percentage of macrophages, polymorphonuclear neutrophils, and dendritic cells were detected by flow cytometry in the infected WT and Trem2 −/− corneas at 5 days post infection. Data were shown to represent one of three individual experiments each with five mice per group.

    Journal: Frontiers in Immunology

    Article Title: Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis

    doi: 10.3389/fimmu.2018.01121

    Figure Lengend Snippet: Triggering receptors expressed on myeloid cells 2 (TREM-2) inhibited pyroptosis of infiltrating cells in Pseudomonas aeruginosa keratitis. Pyroptosis in the infected cornea assessed with terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining. TUNEL-positive staining (green) was detected in Trem2 −/− (A) versus wild type (WT) (B) corneas at 5 days after infection. Cell nuclei were stained with 4,6-diamino-2-phenyl indole (DAPI; blue). Magnification was 200 and 400× respectively. (C) The protein levels of GSDMD and its N-terminal domain in Trem2 −/− versus WT B6 corneas were detected with western blot. (D) The percentage of macrophages, polymorphonuclear neutrophils, and dendritic cells were detected by flow cytometry in the infected WT and Trem2 −/− corneas at 5 days post infection. Data were shown to represent one of three individual experiments each with five mice per group.

    Article Snippet: P. aeruginosa (ATCC 19660) was killed by heating 100 μl aliquots of bacteria solution [108 colony forming units (CFU)/ml] at 65°C for an hour.

    Techniques: Infection, End Labeling, TUNEL Assay, Staining, Western Blot, Flow Cytometry, Cytometry, Mouse Assay

    Triggering receptors expressed on myeloid cells 2 (TREM-2) decreased corneal inflammation and bacterial load in Pseudomonas aeruginosa keratitis via inhibiting caspase-1. Wild type (WT) mice were subconjunctivally injected with the vehicle control (DMSO) and Trem2 −/− mice were subconjunctivally injected with the vehicle control (DMSO) or caspase-1 inhibitor YVAD, and then infected with P. aeruginosa routinely. (A) Clinical score was recorded for each cornea at 1 and 5 days after infection. (B) Representative photographs of infected eyes in YVAD- versus DMSO-treated Trem2 −/− mice as well as DMSO-treated WT mice were taken at 1 and 5 days after infection. (C) Bacterial load in the infected corneas was examined by plate count in YVAD- versus DMSO-treated Trem2 −/− corneas as well as DMSO-treated WT corneas at 1 and 5 days after infection. (D) Hematoxylin and eosin staining was used to examine the histopathology of infected eyes in DMSO-treated WT (left), DMSO-treated Trem 2 −/− (middle), and YVAD-treated Trem 2 −/− (right) mice at 1 and 5 days after infection. Magnification = 100×. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (A,B) or five mice per group (C,D) . * P

    Journal: Frontiers in Immunology

    Article Title: Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis

    doi: 10.3389/fimmu.2018.01121

    Figure Lengend Snippet: Triggering receptors expressed on myeloid cells 2 (TREM-2) decreased corneal inflammation and bacterial load in Pseudomonas aeruginosa keratitis via inhibiting caspase-1. Wild type (WT) mice were subconjunctivally injected with the vehicle control (DMSO) and Trem2 −/− mice were subconjunctivally injected with the vehicle control (DMSO) or caspase-1 inhibitor YVAD, and then infected with P. aeruginosa routinely. (A) Clinical score was recorded for each cornea at 1 and 5 days after infection. (B) Representative photographs of infected eyes in YVAD- versus DMSO-treated Trem2 −/− mice as well as DMSO-treated WT mice were taken at 1 and 5 days after infection. (C) Bacterial load in the infected corneas was examined by plate count in YVAD- versus DMSO-treated Trem2 −/− corneas as well as DMSO-treated WT corneas at 1 and 5 days after infection. (D) Hematoxylin and eosin staining was used to examine the histopathology of infected eyes in DMSO-treated WT (left), DMSO-treated Trem 2 −/− (middle), and YVAD-treated Trem 2 −/− (right) mice at 1 and 5 days after infection. Magnification = 100×. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (A,B) or five mice per group (C,D) . * P

    Article Snippet: P. aeruginosa (ATCC 19660) was killed by heating 100 μl aliquots of bacteria solution [108 colony forming units (CFU)/ml] at 65°C for an hour.

    Techniques: Mouse Assay, Injection, Infection, Staining, Histopathology

    Triggering receptors expressed on myeloid cells 2 (TREM-2) promoted host resistance against Pseudomonas aeruginosa keratitis. Trem2 −/− and wild type (WT) C57BL/6 mice were infected with P. aeruginosa . (A) Clinical score was recorded for each cornea at 1, 3, and 5 days after infection. (B) Representative slit-lamp photographs of infected eyes in WT (left) and Trem2 −/− (right) mice were taken 1, 3, and 5 days after infection. (C) Bacterial load in the infected corneas was examined by plate count assay in WT versus Trem2 −/− mice at 1 and 5 days after infection. (D) Hematoxylin and eosin staining was used to examine the histopathology of infected eyes in WT (left) and Trem2 −/− (right) mice at 1 and 5 days after infection. Magnification = 100×. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (A,B) or five mice per group (C,D) . * P

    Journal: Frontiers in Immunology

    Article Title: Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis

    doi: 10.3389/fimmu.2018.01121

    Figure Lengend Snippet: Triggering receptors expressed on myeloid cells 2 (TREM-2) promoted host resistance against Pseudomonas aeruginosa keratitis. Trem2 −/− and wild type (WT) C57BL/6 mice were infected with P. aeruginosa . (A) Clinical score was recorded for each cornea at 1, 3, and 5 days after infection. (B) Representative slit-lamp photographs of infected eyes in WT (left) and Trem2 −/− (right) mice were taken 1, 3, and 5 days after infection. (C) Bacterial load in the infected corneas was examined by plate count assay in WT versus Trem2 −/− mice at 1 and 5 days after infection. (D) Hematoxylin and eosin staining was used to examine the histopathology of infected eyes in WT (left) and Trem2 −/− (right) mice at 1 and 5 days after infection. Magnification = 100×. Data are the mean ± SEM and represent three individual experiments each with 10 mice per group (A,B) or five mice per group (C,D) . * P

    Article Snippet: P. aeruginosa (ATCC 19660) was killed by heating 100 μl aliquots of bacteria solution [108 colony forming units (CFU)/ml] at 65°C for an hour.

    Techniques: Mouse Assay, Infection, Staining, Histopathology

    OPLS-DA analysis of TTGE profiles, according to disease severity. Panel A) On the left are reported representative TTGE profiles from severe and mild CF patients. Simca-P+ software was used to compute weight coefficients for each TTGE band (87 x variables) on the two subgroups of CF patients (2 y variables) harbouring at least one F508del mutation in one allele: severe (S) and mild (M), with a scaled a centred data set. A clustered image heatmap was generated with CIMminer online software (panel A, right). As depicted in the color-coded legend, the higher that the coefficient value is, the higher the weight (red), while the lower that the value is, the lower the weight (turquoise). Panel B) Faecal TTGE profiles of all 24 CF patients harbouring at least one F508del allele were analysed by OPLS-DA, and the resulting 3D score plot model gave a significant separation among the two sub-groups: severe (S, black diamonds) and mild (M, red diamonds).

    Journal: PLoS ONE

    Article Title: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients

    doi: 10.1371/journal.pone.0061176

    Figure Lengend Snippet: OPLS-DA analysis of TTGE profiles, according to disease severity. Panel A) On the left are reported representative TTGE profiles from severe and mild CF patients. Simca-P+ software was used to compute weight coefficients for each TTGE band (87 x variables) on the two subgroups of CF patients (2 y variables) harbouring at least one F508del mutation in one allele: severe (S) and mild (M), with a scaled a centred data set. A clustered image heatmap was generated with CIMminer online software (panel A, right). As depicted in the color-coded legend, the higher that the coefficient value is, the higher the weight (red), while the lower that the value is, the lower the weight (turquoise). Panel B) Faecal TTGE profiles of all 24 CF patients harbouring at least one F508del allele were analysed by OPLS-DA, and the resulting 3D score plot model gave a significant separation among the two sub-groups: severe (S, black diamonds) and mild (M, red diamonds).

    Article Snippet: Three-dimensional score plots were generated on a presence/absence TTGE matrix data by means of PLS-DA/OPLS-DA algorithms implemented in Simca-P+ software (Umetrics, Umeå, Sweden), taking into account two principal components and one orthogonal component.

    Techniques: Software, Mutagenesis, Generated

    PLS-DA analysis of TTGE profiles, according to F508del mutation. Panel A) On the left are reported representative TTGE profiles from homozygous-F508del (Hm), heterozygous-F508del (Ht) and non-F508del (N) patients. Simca-P+ software was used to compute weight coefficients for each TTGE band (87 x variables) on the three different sub-groups of CF patients (3 y variables, Hm-Ht-N), with a scaled and centred data set. These coefficients were useful to interpret the influence of the x variables on the y ones. A clustered image heatmap was generated with CIMminer online software (panel A, right). As depicted in the color-coded legend, the higher that the coefficient value is, the higher the weight (red), while the lower that the value is, the lower the weight (turquoise). Panel B) Faecal TTGE profiles of all 36 CF patients were analysed by PLS-DA, and the resulting 3D score plot model gave a significant separation between the three sub-groups: homozygous-F508del (Hm, red circles); heterozygous-F508del (Ht, black circles), and non-F508del (N, blue circles).

    Journal: PLoS ONE

    Article Title: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients

    doi: 10.1371/journal.pone.0061176

    Figure Lengend Snippet: PLS-DA analysis of TTGE profiles, according to F508del mutation. Panel A) On the left are reported representative TTGE profiles from homozygous-F508del (Hm), heterozygous-F508del (Ht) and non-F508del (N) patients. Simca-P+ software was used to compute weight coefficients for each TTGE band (87 x variables) on the three different sub-groups of CF patients (3 y variables, Hm-Ht-N), with a scaled and centred data set. These coefficients were useful to interpret the influence of the x variables on the y ones. A clustered image heatmap was generated with CIMminer online software (panel A, right). As depicted in the color-coded legend, the higher that the coefficient value is, the higher the weight (red), while the lower that the value is, the lower the weight (turquoise). Panel B) Faecal TTGE profiles of all 36 CF patients were analysed by PLS-DA, and the resulting 3D score plot model gave a significant separation between the three sub-groups: homozygous-F508del (Hm, red circles); heterozygous-F508del (Ht, black circles), and non-F508del (N, blue circles).

    Article Snippet: Three-dimensional score plots were generated on a presence/absence TTGE matrix data by means of PLS-DA/OPLS-DA algorithms implemented in Simca-P+ software (Umetrics, Umeå, Sweden), taking into account two principal components and one orthogonal component.

    Techniques: Mutagenesis, Software, Generated

    The influence of exonuclease activity on rNMP incorporation by Pol γ. ( A ) Schematic view of reaction set up in Fig 3B. ( B ) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo - ) Pol γ. In vitro replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α- 32 P]-dCTP. Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. ( C ) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo - (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. ( D ) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT PolgA (n = 2) and exonuclease-deficient PolgA D257A (n = 3; “exo - ”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). ( E ) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT PolgA and PolgA D257A mice.

    Journal: PLoS Genetics

    Article Title: The presence of rNTPs decreases the speed of mitochondrial DNA replication

    doi: 10.1371/journal.pgen.1007315

    Figure Lengend Snippet: The influence of exonuclease activity on rNMP incorporation by Pol γ. ( A ) Schematic view of reaction set up in Fig 3B. ( B ) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo - ) Pol γ. In vitro replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α- 32 P]-dCTP. Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. ( C ) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo - (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. ( D ) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT PolgA (n = 2) and exonuclease-deficient PolgA D257A (n = 3; “exo - ”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). ( E ) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT PolgA and PolgA D257A mice.

    Article Snippet: To follow the reaction, [α-32 P]-dCTP (Perkin Elmer) was added.

    Techniques: Activity Assay, In Vitro, Southern Blot, Isolation, Mouse Assay

    rNMP incorporation frequency of Pol γ and the mtDNA replisome on long DNA templates. ( A ) Schematic diagram of the 7.3 kb M13 ssDNA substrate used to compare the incorporation of rNMPs by Pol γ and yeast Pol δ in the primer extension assay in Fig 2B. The newly synthesized DNA was labelled by addition of [α- 32 P]-dCTP to the reaction. ( B ) Analysis of the rNMP incorporation frequency of Pol γ and yeast Pol δ at “normal” (“N”, S phase) concentrations, “low” (“L”, concentration during the rest of the cell cycle and in non-dividing cells), or S . cerevisiae (“Sc”) dNTP concentrations, in the absence or presence of rNTPs. In all reactions, the DNA template was coated with the relevant single-stranded DNA-binding proteins (lanes 1–6 with RPA; lanes 7–14 with mtSSB) to avoid stalling of DNA synthesis due to formation of DNA secondary structures. Untreated (“-NaOH”) or alkaline (“+NaOH”) treated reaction products were analysed on an agarose gel under denaturing conditions. To estimate rNMP incorporation frequencies from the presented gel, the median length of alkali stable products was determined and used to calculate the frequencies as described in Materials and Methods. Numbers on the left-hand side of the gel indicate positions of DNA marker bands and the full-length starting product (7.3) in kb. The gel is a representative picture of four independent experiments. ( C ) Schematic diagram of the primed mini-circle substrate with a 40 nt 5′ overhang used in Fig 2D. ( D ) Analysis of the rNMP incorporation frequency by the mitochondrial replisome consisting of mtSSB, Twinkle and Pol γ (AB 2 ) on a primed mini-circle substrate with a 5′ overhang for Twinkle loading. Reactions were carried out at normal (“N), low (“L”), and “ S . cerevisiae ” (“Sc”) dNTP concentrations in the presence or absence of rNTPs. Untreated (“-NaOH”) and alkaline (“+NaOH”) treated reaction products were analysed on a denaturing (alkaline) agarose gel. The gel is a representative picture of two independent experiments.

    Journal: PLoS Genetics

    Article Title: The presence of rNTPs decreases the speed of mitochondrial DNA replication

    doi: 10.1371/journal.pgen.1007315

    Figure Lengend Snippet: rNMP incorporation frequency of Pol γ and the mtDNA replisome on long DNA templates. ( A ) Schematic diagram of the 7.3 kb M13 ssDNA substrate used to compare the incorporation of rNMPs by Pol γ and yeast Pol δ in the primer extension assay in Fig 2B. The newly synthesized DNA was labelled by addition of [α- 32 P]-dCTP to the reaction. ( B ) Analysis of the rNMP incorporation frequency of Pol γ and yeast Pol δ at “normal” (“N”, S phase) concentrations, “low” (“L”, concentration during the rest of the cell cycle and in non-dividing cells), or S . cerevisiae (“Sc”) dNTP concentrations, in the absence or presence of rNTPs. In all reactions, the DNA template was coated with the relevant single-stranded DNA-binding proteins (lanes 1–6 with RPA; lanes 7–14 with mtSSB) to avoid stalling of DNA synthesis due to formation of DNA secondary structures. Untreated (“-NaOH”) or alkaline (“+NaOH”) treated reaction products were analysed on an agarose gel under denaturing conditions. To estimate rNMP incorporation frequencies from the presented gel, the median length of alkali stable products was determined and used to calculate the frequencies as described in Materials and Methods. Numbers on the left-hand side of the gel indicate positions of DNA marker bands and the full-length starting product (7.3) in kb. The gel is a representative picture of four independent experiments. ( C ) Schematic diagram of the primed mini-circle substrate with a 40 nt 5′ overhang used in Fig 2D. ( D ) Analysis of the rNMP incorporation frequency by the mitochondrial replisome consisting of mtSSB, Twinkle and Pol γ (AB 2 ) on a primed mini-circle substrate with a 5′ overhang for Twinkle loading. Reactions were carried out at normal (“N), low (“L”), and “ S . cerevisiae ” (“Sc”) dNTP concentrations in the presence or absence of rNTPs. Untreated (“-NaOH”) and alkaline (“+NaOH”) treated reaction products were analysed on a denaturing (alkaline) agarose gel. The gel is a representative picture of two independent experiments.

    Article Snippet: To follow the reaction, [α-32 P]-dCTP (Perkin Elmer) was added.

    Techniques: Primer Extension Assay, Synthesized, Concentration Assay, DNA Binding Assay, Recombinase Polymerase Amplification, DNA Synthesis, Agarose Gel Electrophoresis, Marker

    The anti-inflammatory and anti-fibrotic effects of 5-MTP in vitro. a , b The protein expression and relative quantitative data of IκBα, p-IκBα, NF-κB p65, COX-2 and MCP-1 as well as Nrf2, HO-1 and NQO-1 in HK-2 cells induced by TGF-β1 as indicated. * P

    Journal: Nature Communications

    Article Title: Identification of serum metabolites associating with chronic kidney disease progression and anti-fibrotic effect of 5-methoxytryptophan

    doi: 10.1038/s41467-019-09329-0

    Figure Lengend Snippet: The anti-inflammatory and anti-fibrotic effects of 5-MTP in vitro. a , b The protein expression and relative quantitative data of IκBα, p-IκBα, NF-κB p65, COX-2 and MCP-1 as well as Nrf2, HO-1 and NQO-1 in HK-2 cells induced by TGF-β1 as indicated. * P

    Article Snippet: The following primary antibodies were employed (dilution): collagen I (1:5000, ab34710, Abcam, USA), α-SMA (1:300, ab7817, Abcam, USA), fibronectin (1:1000, ab2413, Abcam, USA), vimentin (1:1000, ab92547, Abcam, USA), E-cadherin (1:500, ab76055, Abcam, USA), p-IκBα (1:2000, 2859, Cell Signaling Technology, USA), IκBα (1:2000, 4812, Cell Signaling Technology, USA), NF-κB p65 (1:1000, ab16502, Abcam, USA), MCP-1 (1:1000, ab7202, Abcam, USA), COX-2 (1:1000, ab62331, Abcam, USA), Nrf2 (1:1000, ab31163, Abcam, USA), HO-1 (1:1000, ab68477, Abcam, USA), NQO-1 (1:1000, ab28947, Abcam, USA) and TPH-1 (1:500, ab52954, Abcam, USA).

    Techniques: In Vitro, Expressing