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    Structured Review

    Thermo Fisher genes has1 3
    Factor analysis for correlation of gene expression levels. <t>HAS1-3</t> , HYAL1-2 , CEMIP , CD44 , VCAN , and TSG6 in non-failing left wall and septum (n = 9) ( a ) and HCM myectomies (n = 5) ( b ). ( a ) In left wall and septum there are two clusters where HAS1 , CEMIP , CD44 , VCAN , and TSG6 form one cluster, and HAS2 , HAS3, and HYAL2 form another. ( b ) In basal septal myectomies from HCM patients the expression levels of CEMIP , CD44, and VCAN formed a new correlation cluster with HAS3 . HAS2 , HYAL1 , and TSG6 formed another cluster. The levels of HAS1 and HYAL2 no longer correlated with any of the other genes investigated. Factor analysis was performed with the principal components method to analyze the correlation matrix and two factors were extracted.
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    Images

    1) Product Images from "Low Molecular Mass Myocardial Hyaluronan in Human Hypertrophic Cardiomyopathy"

    Article Title: Low Molecular Mass Myocardial Hyaluronan in Human Hypertrophic Cardiomyopathy

    Journal: Cells

    doi: 10.3390/cells8020097

    Factor analysis for correlation of gene expression levels. HAS1-3 , HYAL1-2 , CEMIP , CD44 , VCAN , and TSG6 in non-failing left wall and septum (n = 9) ( a ) and HCM myectomies (n = 5) ( b ). ( a ) In left wall and septum there are two clusters where HAS1 , CEMIP , CD44 , VCAN , and TSG6 form one cluster, and HAS2 , HAS3, and HYAL2 form another. ( b ) In basal septal myectomies from HCM patients the expression levels of CEMIP , CD44, and VCAN formed a new correlation cluster with HAS3 . HAS2 , HYAL1 , and TSG6 formed another cluster. The levels of HAS1 and HYAL2 no longer correlated with any of the other genes investigated. Factor analysis was performed with the principal components method to analyze the correlation matrix and two factors were extracted.
    Figure Legend Snippet: Factor analysis for correlation of gene expression levels. HAS1-3 , HYAL1-2 , CEMIP , CD44 , VCAN , and TSG6 in non-failing left wall and septum (n = 9) ( a ) and HCM myectomies (n = 5) ( b ). ( a ) In left wall and septum there are two clusters where HAS1 , CEMIP , CD44 , VCAN , and TSG6 form one cluster, and HAS2 , HAS3, and HYAL2 form another. ( b ) In basal septal myectomies from HCM patients the expression levels of CEMIP , CD44, and VCAN formed a new correlation cluster with HAS3 . HAS2 , HYAL1 , and TSG6 formed another cluster. The levels of HAS1 and HYAL2 no longer correlated with any of the other genes investigated. Factor analysis was performed with the principal components method to analyze the correlation matrix and two factors were extracted.

    Techniques Used: Expressing

    2) Product Images from "Catalytic Intermediates of Inducible Nitric-oxide Synthase Stabilized by the W188H Mutation *"

    Article Title: Catalytic Intermediates of Inducible Nitric-oxide Synthase Stabilized by the W188H Mutation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.403238

    Resonance Raman spectra of the ferric derivatives of the W188H mutant of iNOS oxy in the low frequency ( a ) and high frequency window ( b ). The laser excitation was 413.1 nm.
    Figure Legend Snippet: Resonance Raman spectra of the ferric derivatives of the W188H mutant of iNOS oxy in the low frequency ( a ) and high frequency window ( b ). The laser excitation was 413.1 nm.

    Techniques Used: Mutagenesis

    3) Product Images from "An improved LC-MS-MS method for the quantification of prostaglandins E2 and D2 production in biological fluids"

    Article Title: An improved LC-MS-MS method for the quantification of prostaglandins E2 and D2 production in biological fluids

    Journal:

    doi: 10.1016/j.ab.2007.08.041

    Negative ion electrospray product ion tandem mass spectra of PGE 2 and PGD 2 (10 ng/mL).
    Figure Legend Snippet: Negative ion electrospray product ion tandem mass spectra of PGE 2 and PGD 2 (10 ng/mL).

    Techniques Used:

    Negative ion electrospray LC-MS-MS chromatograms obtained using reversed phase HPLC and collision-induced dissociation with MRM of PGE 2 , PGD 2 and d 4 -PGE 2 . A) PGE 2 and PGD 2 standards at 10 ng/mL (28.4 nM); B) internal standards d 4 -PGE 2 and d 4 -PGD 2 at 10
    Figure Legend Snippet: Negative ion electrospray LC-MS-MS chromatograms obtained using reversed phase HPLC and collision-induced dissociation with MRM of PGE 2 , PGD 2 and d 4 -PGE 2 . A) PGE 2 and PGD 2 standards at 10 ng/mL (28.4 nM); B) internal standards d 4 -PGE 2 and d 4 -PGD 2 at 10

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, High Performance Liquid Chromatography

    4) Product Images from "Statin Treatment Increases Lifespan and Improves Cardiac Health in Drosophila by Decreasing Specific Protein Prenylation"

    Article Title: Statin Treatment Increases Lifespan and Improves Cardiac Health in Drosophila by Decreasing Specific Protein Prenylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039581

    Simvastatin administration to mice decreased prenylation of specific small G proteins, as measured by a decrease in their membrane association. Panel A, Western blot showing the level of Rab4, Ras, calnexin, and α-tubulin in the cytoplasmic and membrane fractions purified from the liver of control and simvastatin treated mice. Panel B shows the quantification of the data shown in Panel A. The levels of Rab4 in the cytoplasmic fractions were normalized to the level of α-tubulin in each sample. The levels of Rab 4 in the membrane fractions were normalized to the level of calnexin in each sample. Means and standard errors obtained with tissue from control (open bars) and simvastatin treated (closed bars) mice are shown. One of the precipitated membrane samples was overloaded on this blot, and the amount of Rab4 reported in panel B was determined from another blot. The scanner units were adjusted to facilitate comparisons between membrane and cytoplasmic fractions.
    Figure Legend Snippet: Simvastatin administration to mice decreased prenylation of specific small G proteins, as measured by a decrease in their membrane association. Panel A, Western blot showing the level of Rab4, Ras, calnexin, and α-tubulin in the cytoplasmic and membrane fractions purified from the liver of control and simvastatin treated mice. Panel B shows the quantification of the data shown in Panel A. The levels of Rab4 in the cytoplasmic fractions were normalized to the level of α-tubulin in each sample. The levels of Rab 4 in the membrane fractions were normalized to the level of calnexin in each sample. Means and standard errors obtained with tissue from control (open bars) and simvastatin treated (closed bars) mice are shown. One of the precipitated membrane samples was overloaded on this blot, and the amount of Rab4 reported in panel B was determined from another blot. The scanner units were adjusted to facilitate comparisons between membrane and cytoplasmic fractions.

    Techniques Used: Mouse Assay, Western Blot, Purification

    Juvenile hormone signaling and simvastatin treatment stimulate lifespan synergistically. Shown are the lifespans of Drosophila in the absence of drugs [control (○)]; and in the presence of 240 µM simvastatin (□); 320 µM methoprene (▵); and 320 µM methoprene and 240 µM simvastatin together (▾). The mean lifespan of the simvastatin, methoprene, and simvastatin with methoprene treated flies was significantly increased (P = 0.02, P = 0.0034, and P
    Figure Legend Snippet: Juvenile hormone signaling and simvastatin treatment stimulate lifespan synergistically. Shown are the lifespans of Drosophila in the absence of drugs [control (○)]; and in the presence of 240 µM simvastatin (□); 320 µM methoprene (▵); and 320 µM methoprene and 240 µM simvastatin together (▾). The mean lifespan of the simvastatin, methoprene, and simvastatin with methoprene treated flies was significantly increased (P = 0.02, P = 0.0034, and P

    Techniques Used:

    Simvastatin extends the lifespan of Drosophila . Simvastatin was fed to the flies in their food beginning on the first day of eclosure. Shown is survival at zero (○), 0.024 (□), 0.24 (▵), 2.4 (▿), and 12 (⋄) mM simvastatin. As judged by the log rank test, the lifespan of the flies was significantly changed when they were treated with 24 µM (P = 0.047), 240 µM (P
    Figure Legend Snippet: Simvastatin extends the lifespan of Drosophila . Simvastatin was fed to the flies in their food beginning on the first day of eclosure. Shown is survival at zero (○), 0.024 (□), 0.24 (▵), 2.4 (▿), and 12 (⋄) mM simvastatin. As judged by the log rank test, the lifespan of the flies was significantly changed when they were treated with 24 µM (P = 0.047), 240 µM (P

    Techniques Used:

    Simvastatin administration reduces the number of old flies that exhibit severe arrhythmias, as quantified by the AI, a measure of heartbeat regularity. Panel A shows a summary of the results showing that AI is significantly reduced in hearts from 4 week old flies fed simvastatin compared to vehicle (n = 35 flies for each group; p
    Figure Legend Snippet: Simvastatin administration reduces the number of old flies that exhibit severe arrhythmias, as quantified by the AI, a measure of heartbeat regularity. Panel A shows a summary of the results showing that AI is significantly reduced in hearts from 4 week old flies fed simvastatin compared to vehicle (n = 35 flies for each group; p

    Techniques Used:

    Ubiquinone administration slightly reduces lifespan. Shown are the lifespan of flies consuming food containing no drugs [control (○)]; 240 µM simvastatin (□); 5 mM CoQ10 (▾); 5 mM CoQ10 and 240 µM simvastatin (•). The mean lifespans of the flies treated with simvastatin alone (P
    Figure Legend Snippet: Ubiquinone administration slightly reduces lifespan. Shown are the lifespan of flies consuming food containing no drugs [control (○)]; 240 µM simvastatin (□); 5 mM CoQ10 (▾); 5 mM CoQ10 and 240 µM simvastatin (•). The mean lifespans of the flies treated with simvastatin alone (P

    Techniques Used:

    Supplementation with simvastatin and CoQ10 reduces endogenous ubiquinone levels. Ubiquinone (5 mM) and simvastatin (240 µM) administered to Drosophila in their food alone or together decreased the endogenous levels of CoQ10 as measured by LC-MS/MS. Two asterisks indicate the differences were highly significant (P≤0.01) and three indicate that the differences were very highly significant (P≤0.001). Sim below a bar indicates the flies were treated with simvastatin.
    Figure Legend Snippet: Supplementation with simvastatin and CoQ10 reduces endogenous ubiquinone levels. Ubiquinone (5 mM) and simvastatin (240 µM) administered to Drosophila in their food alone or together decreased the endogenous levels of CoQ10 as measured by LC-MS/MS. Two asterisks indicate the differences were highly significant (P≤0.01) and three indicate that the differences were very highly significant (P≤0.001). Sim below a bar indicates the flies were treated with simvastatin.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    5) Product Images from "Modified Seed Growth of Iron Oxide Nanoparticles in Benzyl Alcohol — Optimization for Heating and Broad Stability in Biomedical Applications"

    Article Title: Modified Seed Growth of Iron Oxide Nanoparticles in Benzyl Alcohol — Optimization for Heating and Broad Stability in Biomedical Applications

    Journal: Nanobiomedicine

    doi: 10.5772/60035

    Heating curves of 3 mL of water and iron oxide samples dispersed in 0.25% TMAOH. Concentrations of iron determined by Prussian Blue UV-VIS are 0, 14.48, 15.40, 14.00, and 15.36 mg/mL for deionized water (blue), A2–24 under N2 (green), A2–24 (red), A2–24_B2–24 (purple), and A2–24(195)_B2–24(195) (black) respectively. An alternating magnetic field 175.4 A at frequency of 270 kHz for 600 seconds was used, and the temperature was recorded every 1.4 seconds.
    Figure Legend Snippet: Heating curves of 3 mL of water and iron oxide samples dispersed in 0.25% TMAOH. Concentrations of iron determined by Prussian Blue UV-VIS are 0, 14.48, 15.40, 14.00, and 15.36 mg/mL for deionized water (blue), A2–24 under N2 (green), A2–24 (red), A2–24_B2–24 (purple), and A2–24(195)_B2–24(195) (black) respectively. An alternating magnetic field 175.4 A at frequency of 270 kHz for 600 seconds was used, and the temperature was recorded every 1.4 seconds.

    Techniques Used:

    6) Product Images from "Deep Phosphoproteomic Measurements Pinpointing Drug Induced Protective Mechanisms in Neuronal Cells"

    Article Title: Deep Phosphoproteomic Measurements Pinpointing Drug Induced Protective Mechanisms in Neuronal Cells

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2016.00635

    The fractionated phosphopeptides were analyzed by an EASY n-LC 1000 system directly connected to a Q Exactive (QE) or a Q Exactive HF (HF). (A)  Distribution of phosphoproteins identified in cells. Identified phosphorylation proteins were assigned to DAVID GO terms for cellular component. Numbers of proteins belonging to each GO term are demonstrated as percentages of the total number of proteins. GO terms significantly over- and under-represented are marked [fold enrichment ≥ 1.4 or ≤ 0.67,  P  ≤ 0.01 ( ** )].  (B)  Function classification of phosphoproteins identified. The sorting was done with Ingenuity IPA and the bar plot was generated with GraphPad.  (C)  Scatter plots of the log-transformed ratios Aβ 25−35 /Control (Ab vs. Con), (Aβ 25−35 +GFKP-19)/Control (GF vs. Con), and (Aβ 25−35 +GFKP-19)/Aβ 25−35  (GF vs. Ab) demonstrate the correlation between QE results and HF results. Black dash lines represent 1.5 FC. The correlation coefficient (R) were calculated using spearman.
    Figure Legend Snippet: The fractionated phosphopeptides were analyzed by an EASY n-LC 1000 system directly connected to a Q Exactive (QE) or a Q Exactive HF (HF). (A) Distribution of phosphoproteins identified in cells. Identified phosphorylation proteins were assigned to DAVID GO terms for cellular component. Numbers of proteins belonging to each GO term are demonstrated as percentages of the total number of proteins. GO terms significantly over- and under-represented are marked [fold enrichment ≥ 1.4 or ≤ 0.67, P ≤ 0.01 ( ** )]. (B) Function classification of phosphoproteins identified. The sorting was done with Ingenuity IPA and the bar plot was generated with GraphPad. (C) Scatter plots of the log-transformed ratios Aβ 25−35 /Control (Ab vs. Con), (Aβ 25−35 +GFKP-19)/Control (GF vs. Con), and (Aβ 25−35 +GFKP-19)/Aβ 25−35 (GF vs. Ab) demonstrate the correlation between QE results and HF results. Black dash lines represent 1.5 FC. The correlation coefficient (R) were calculated using spearman.

    Techniques Used: Indirect Immunoperoxidase Assay, Generated, Transformation Assay

    7) Product Images from "Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms"

    Article Title: Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms

    Journal: mBio

    doi: 10.1128/mBio.01593-17

    Targeted proteomic analysis of a biofilm subpopulation. (A) Detection of mCherry fluorescence (green) in live biofilms was used to locate cells expressing the NLL-MetRS–mCherry fusion. Biofilms were counterstained with SYTO9 (magenta) immediately before imaging. (B) Following Anl treatment, BONCAT labeling in biofilms was visualized by treating fixed biofilms with DBCO-TAMRA (green). Biofilms were counterstained with SYTO9 (magenta). Colocalization of fluorescent signals is displayed in white. For panels A and B, cross-sections were reconstructed from confocal image stacks. (C) Proteins identified following BONCAT enrichment from P rpoS : nll-mc and P trc : nll-mc strains. (D) Quantification of relative protein abundances following enrichment from both strains. Ribosomal proteins are shown in orange. Proteins discussed in the text are indicated by gene name. The complete set of LFQ values, ratios, and adjusted P values is provided in Data Set S1 . (E) Spatial distribution of GFP expression (green) under control of the rpoS or algP promoters in live biofilms. Biofilms were counterstained with SYTO62 (magenta).
    Figure Legend Snippet: Targeted proteomic analysis of a biofilm subpopulation. (A) Detection of mCherry fluorescence (green) in live biofilms was used to locate cells expressing the NLL-MetRS–mCherry fusion. Biofilms were counterstained with SYTO9 (magenta) immediately before imaging. (B) Following Anl treatment, BONCAT labeling in biofilms was visualized by treating fixed biofilms with DBCO-TAMRA (green). Biofilms were counterstained with SYTO9 (magenta). Colocalization of fluorescent signals is displayed in white. For panels A and B, cross-sections were reconstructed from confocal image stacks. (C) Proteins identified following BONCAT enrichment from P rpoS : nll-mc and P trc : nll-mc strains. (D) Quantification of relative protein abundances following enrichment from both strains. Ribosomal proteins are shown in orange. Proteins discussed in the text are indicated by gene name. The complete set of LFQ values, ratios, and adjusted P values is provided in Data Set S1 . (E) Spatial distribution of GFP expression (green) under control of the rpoS or algP promoters in live biofilms. Biofilms were counterstained with SYTO62 (magenta).

    Techniques Used: Fluorescence, Expressing, Imaging, Labeling

    8) Product Images from "C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae"

    Article Title: C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007341

    C-terminal modification of VesB. VesB was overexpressed and purified from supernatants of LB-grown V . cholerae culture in the presence of protease inhibitors at 4° C. The purified material was subjected to intact mass analysis (A) and SDS-PAGE, in-gel trypsin digestion and LC-MS/MS analysis (B-D). A. Left panel: Deconvoluted ESI mass spectrum indicates four molecular masses of VesB. The masses highlighted with asterisks correspond to theoretical VesB masses generated through cleavage at either Ser 378 ( * ) or Ser 380 ( ** ) (see inset with C-terminal sequence of VesB). The relative amount of the four VesB species varied between three separate purifications. Accuracy of the instrument: 0.01% of molecular mass. Right panel: The deconvoluted mass spectrum of protein eluting at a higher acetonitrile concentration. B. C-terminal peptides of VesB generated by trypsin and identified by LC-MS/MS analysis. The asterisks correspond to cleavage at Ser 378 ( * ) and Ser 380 ( ** ). C. Representative MS/MS spectrum for the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. D. MS/MS spectrum for the VesB peptide IQLDTSPFAPSASSGG with a C-terminal 197 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da). E. VesB was extracted from WT V . cholerae with Triton X-100, purified on benzamidine sepharose and subjected to SDS-PAGE, in-gel trypsin digestion and LC -MS/MS analysis. Representative MS/MS spectrum of the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. F. MS/MS spectrum of the 197-Da modified peptide IQLDTSPFAPSASSGG as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da). G. VesB is attached to a glycerophosphoethanolamine containing moiety, possibly phosphatidylethanolamine, via its C-terminal glycine. The red and blue arrows indicate possible sites of hydrolysis or fragmentation, respectively, resulting in VesB species with either ethanolamine or glycerophosphoethanolamine.
    Figure Legend Snippet: C-terminal modification of VesB. VesB was overexpressed and purified from supernatants of LB-grown V . cholerae culture in the presence of protease inhibitors at 4° C. The purified material was subjected to intact mass analysis (A) and SDS-PAGE, in-gel trypsin digestion and LC-MS/MS analysis (B-D). A. Left panel: Deconvoluted ESI mass spectrum indicates four molecular masses of VesB. The masses highlighted with asterisks correspond to theoretical VesB masses generated through cleavage at either Ser 378 ( * ) or Ser 380 ( ** ) (see inset with C-terminal sequence of VesB). The relative amount of the four VesB species varied between three separate purifications. Accuracy of the instrument: 0.01% of molecular mass. Right panel: The deconvoluted mass spectrum of protein eluting at a higher acetonitrile concentration. B. C-terminal peptides of VesB generated by trypsin and identified by LC-MS/MS analysis. The asterisks correspond to cleavage at Ser 378 ( * ) and Ser 380 ( ** ). C. Representative MS/MS spectrum for the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. D. MS/MS spectrum for the VesB peptide IQLDTSPFAPSASSGG with a C-terminal 197 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da). E. VesB was extracted from WT V . cholerae with Triton X-100, purified on benzamidine sepharose and subjected to SDS-PAGE, in-gel trypsin digestion and LC -MS/MS analysis. Representative MS/MS spectrum of the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. F. MS/MS spectrum of the 197-Da modified peptide IQLDTSPFAPSASSGG as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da). G. VesB is attached to a glycerophosphoethanolamine containing moiety, possibly phosphatidylethanolamine, via its C-terminal glycine. The red and blue arrows indicate possible sites of hydrolysis or fragmentation, respectively, resulting in VesB species with either ethanolamine or glycerophosphoethanolamine.

    Techniques Used: Modification, Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Generated, Sequencing, Concentration Assay

    Secreted and cell-associated VesB are active. A. Schematic representation of prepro-VesB with the N-terminal signal peptide (SP; residues 1–23) in blue, the pro-peptide (PP; residues 24–32) in green, the protease domain (with the catalytic residues His, Asp and Ser) in dark purple, the Ig-fold domain in light purple, and the GlyGly-CTERM extension (382–403) in magenta. B. The primary sequences of the C-terminal portions of the six proteins in V . cholerae containing the GlyGly-CTERM domain are aligned using the EMBL-EBI multiple sequence alignment tool, Clustal Omega ( http://www.ebi.ac.uk/Tools/msa/clustalo/ ). Residues are labeled according to their physiochemical properties. Magenta represents basic residues. Black denotes small and hydrophobic residues, including aromatic amino acids (except tyrosine). Green residues include glycine and amino acids with either hydroxyl-, sulfhydryl-, or amine groups. Aspartic and glutamic acid are shown in blue. C. The ΔvesABC strain containing empty vector (p), pVesB, or pVesB-S221A were grown in LB with 100 μg/mL carbenicillin and 50 μM IPTG for four hours. Serine protease activity against the fluorogenic peptide Boc-Gln-Ala-Arg-AMC was measured in culture supernatants and suspensions of intact cells. Samples from three independent experiments were each analyzed in technical triplicates and the bars represent means +/- standard error. D. The culture supernatants and cell suspensions from C were subjected to SDS-PAGE and immunoblotting with VesB antibodies. Representative blot is shown. E. Culture supernatants of WT and ΔvesABC strains of V . cholerae expressing plasmid-encoded VesB or VesB-S221A grown for four hours in LB with 100 μg/mL carbenicillin and 50 μM IPTG were analyzed by SDS-PAGE and immunoblotting with VesB antibodies. Representative blot is shown.
    Figure Legend Snippet: Secreted and cell-associated VesB are active. A. Schematic representation of prepro-VesB with the N-terminal signal peptide (SP; residues 1–23) in blue, the pro-peptide (PP; residues 24–32) in green, the protease domain (with the catalytic residues His, Asp and Ser) in dark purple, the Ig-fold domain in light purple, and the GlyGly-CTERM extension (382–403) in magenta. B. The primary sequences of the C-terminal portions of the six proteins in V . cholerae containing the GlyGly-CTERM domain are aligned using the EMBL-EBI multiple sequence alignment tool, Clustal Omega ( http://www.ebi.ac.uk/Tools/msa/clustalo/ ). Residues are labeled according to their physiochemical properties. Magenta represents basic residues. Black denotes small and hydrophobic residues, including aromatic amino acids (except tyrosine). Green residues include glycine and amino acids with either hydroxyl-, sulfhydryl-, or amine groups. Aspartic and glutamic acid are shown in blue. C. The ΔvesABC strain containing empty vector (p), pVesB, or pVesB-S221A were grown in LB with 100 μg/mL carbenicillin and 50 μM IPTG for four hours. Serine protease activity against the fluorogenic peptide Boc-Gln-Ala-Arg-AMC was measured in culture supernatants and suspensions of intact cells. Samples from three independent experiments were each analyzed in technical triplicates and the bars represent means +/- standard error. D. The culture supernatants and cell suspensions from C were subjected to SDS-PAGE and immunoblotting with VesB antibodies. Representative blot is shown. E. Culture supernatants of WT and ΔvesABC strains of V . cholerae expressing plasmid-encoded VesB or VesB-S221A grown for four hours in LB with 100 μg/mL carbenicillin and 50 μM IPTG were analyzed by SDS-PAGE and immunoblotting with VesB antibodies. Representative blot is shown.

    Techniques Used: Sequencing, Labeling, Plasmid Preparation, Activity Assay, SDS Page, Expressing

    9) Product Images from "Multifunctional Carbon Aerogels Derived by Sol–Gel Process of Natural Polysaccharides of Different Botanical Origin"

    Article Title: Multifunctional Carbon Aerogels Derived by Sol–Gel Process of Natural Polysaccharides of Different Botanical Origin

    Journal: Materials

    doi: 10.3390/ma10111336

    3D and 2D Fourier transform infrared (FTIR) spectrum maps of gaseous products evolved during thermal decomposition of ( a , b ) the organic aerogel sample based on potato starch (OAGPS) and ( c ) TGA/DTA/DTG profiles for OAGPS, OAGMS (OAG based on maize starch) and OAGRS (OAG based on rice starch) materials.
    Figure Legend Snippet: 3D and 2D Fourier transform infrared (FTIR) spectrum maps of gaseous products evolved during thermal decomposition of ( a , b ) the organic aerogel sample based on potato starch (OAGPS) and ( c ) TGA/DTA/DTG profiles for OAGPS, OAGMS (OAG based on maize starch) and OAGRS (OAG based on rice starch) materials.

    Techniques Used:

    10) Product Images from "ArsAB, a Novel Enzyme from Sporomusa ovata Activates Phenolic Bases for Adenosylcobamide Biosynthesis"

    Article Title: ArsAB, a Novel Enzyme from Sporomusa ovata Activates Phenolic Bases for Adenosylcobamide Biosynthesis

    Journal: Molecular microbiology

    doi: 10.1111/j.1365-2958.2011.07741.x

    Late steps in AdoCbl biosynthesis in S. enterica For simplicity purposes, the corrin ring is represented as a rhomboid, and lower ligands hydroxyl groups or water molecules are not shown. AdoCby, adenosylcobyric acid; AdoCbi-P, adenosylcobinamide-phosphate; AdoCbi-GDP, adenosylcobinamide-GDP; AdoCbl-P, adenosylcobalamin-phosphate; AdoCbl, adenosylcobalamin; AP-P, aminopropanol phosphate; DMB, 5, 6-dimethylbenzimidazole; NaMN, nicotinate mononucleotide. CbiP (AdoCby synthase) and CbiB (AdoCbiP synthetase) catalyze the last two steps of the de novo corrin ring biosynthetic pathway. CobU (AdoCbi kinase/AdoCbi-P guanylyltransferase), CobS (AdoCbl-P synthase), CobT, NaMN:DMB phosphoribosyltransferase, and CobC (AdoCbl-P phosphatase) comprise the nucleotide loop assembly pathway, also known as the late steps. CobA (ATP :Co(I)rrinoid adenosylytransferase) is the housekeeping corrinoid adenosylating enzyme required for de novo corrin ring biosynthesis, and for the salvaging of incomplete corrinoids like cobinamide. The phenolic substrates for ArsAB and the resulting Cbas synthesized using the α-phenolic-ribotide products are highlighted in black and white. Structures of O -glycosidic phenolic ligands are shown next to corresponding cobamide name. Phenolic ligands do not form a coordination bond with the Co ion of the ring.
    Figure Legend Snippet: Late steps in AdoCbl biosynthesis in S. enterica For simplicity purposes, the corrin ring is represented as a rhomboid, and lower ligands hydroxyl groups or water molecules are not shown. AdoCby, adenosylcobyric acid; AdoCbi-P, adenosylcobinamide-phosphate; AdoCbi-GDP, adenosylcobinamide-GDP; AdoCbl-P, adenosylcobalamin-phosphate; AdoCbl, adenosylcobalamin; AP-P, aminopropanol phosphate; DMB, 5, 6-dimethylbenzimidazole; NaMN, nicotinate mononucleotide. CbiP (AdoCby synthase) and CbiB (AdoCbiP synthetase) catalyze the last two steps of the de novo corrin ring biosynthetic pathway. CobU (AdoCbi kinase/AdoCbi-P guanylyltransferase), CobS (AdoCbl-P synthase), CobT, NaMN:DMB phosphoribosyltransferase, and CobC (AdoCbl-P phosphatase) comprise the nucleotide loop assembly pathway, also known as the late steps. CobA (ATP :Co(I)rrinoid adenosylytransferase) is the housekeeping corrinoid adenosylating enzyme required for de novo corrin ring biosynthesis, and for the salvaging of incomplete corrinoids like cobinamide. The phenolic substrates for ArsAB and the resulting Cbas synthesized using the α-phenolic-ribotide products are highlighted in black and white. Structures of O -glycosidic phenolic ligands are shown next to corresponding cobamide name. Phenolic ligands do not form a coordination bond with the Co ion of the ring.

    Techniques Used: Synthesized

    11) Product Images from "MICELLAR NANOMEDICINE OF HUMAN NEUROPEPTIDE Y"

    Article Title: MICELLAR NANOMEDICINE OF HUMAN NEUROPEPTIDE Y

    Journal: Nanomedicine : nanotechnology, biology, and medicine

    doi: 10.1016/j.nano.2011.01.004

    Representative MALDI-TOF mass spectra of ( A ) NPY (10 μM) in saline (Sal-NPY), ( B ) Sal-NPY + DPP-4 (1 nM), ( C ) NPY-SSM (300 μM DSPE-PEG 2000 ) ( D ) and NPY-SSM + DPP-4. Samples were incubated at 37 °C for 1 h in PBS (pH 7.4) prior
    Figure Legend Snippet: Representative MALDI-TOF mass spectra of ( A ) NPY (10 μM) in saline (Sal-NPY), ( B ) Sal-NPY + DPP-4 (1 nM), ( C ) NPY-SSM (300 μM DSPE-PEG 2000 ) ( D ) and NPY-SSM + DPP-4. Samples were incubated at 37 °C for 1 h in PBS (pH 7.4) prior

    Techniques Used: Incubation

    12) Product Images from "IR-MALDESI mass spectrometry imaging at 50 micron spatial resolution"

    Article Title: IR-MALDESI mass spectrometry imaging at 50 micron spatial resolution

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-017-1740-x

    A) IR-MALDESI MSI of cholesterol [M-H 2 O+H] + , m/z 369.3516 ± 2.5 ppm in hen ovary tissue at 50, 75, and 100 μm spatial resolution. B) An optical image of a stained serial section showing the complex morphology of the ovary tissue. C) An averaged mass spectrum for the 50 μm spatial resolution experiment.
    Figure Legend Snippet: A) IR-MALDESI MSI of cholesterol [M-H 2 O+H] + , m/z 369.3516 ± 2.5 ppm in hen ovary tissue at 50, 75, and 100 μm spatial resolution. B) An optical image of a stained serial section showing the complex morphology of the ovary tissue. C) An averaged mass spectrum for the 50 μm spatial resolution experiment.

    Techniques Used: Staining

    Schematic of IR-MALDESI using A) a single spherical focusing lens and B) a beam expander with an iris and an aspherical focusing lens.
    Figure Legend Snippet: Schematic of IR-MALDESI using A) a single spherical focusing lens and B) a beam expander with an iris and an aspherical focusing lens.

    Techniques Used:

    13) Product Images from "Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase"

    Article Title: Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02253-1

    ARH3 mainly hydrolyzes ADP-ribosylated serines in vitro. a Number of ADP-ribosylated peptide spectra matches (PSMs) or demodified peptide spectra matches after PARG or ARH3 treatment. Data represent means ± SEM for n = 3 independent demodification experiments. b Venn diagrams of unique ADPr peptides and proteins. c Volcano plot of ARH3- and PARG-treated samples. “Unmodified peptides” are shown as open circles and “ADP-ribosylated peptides” as filled circles. ADP-ribosylation sites confirmed by EThcD spectra are annotated and color coded in red as S-ADPr and in blue as R-ADPr sites. ADP-ribosylated peptides with uncertain ADP-ribosylation site localization are shown in black. The black hyperbolic line represents a permutation-based false discovery rate (FDR) of 5% and a minimal fold change of 2. d Normalized abundance of individual Ser- and Arg-ADPr peptides after PARG or ARH3 treatment. Data represent means ± SEM for n = 3 independent demodification experiments
    Figure Legend Snippet: ARH3 mainly hydrolyzes ADP-ribosylated serines in vitro. a Number of ADP-ribosylated peptide spectra matches (PSMs) or demodified peptide spectra matches after PARG or ARH3 treatment. Data represent means ± SEM for n = 3 independent demodification experiments. b Venn diagrams of unique ADPr peptides and proteins. c Volcano plot of ARH3- and PARG-treated samples. “Unmodified peptides” are shown as open circles and “ADP-ribosylated peptides” as filled circles. ADP-ribosylation sites confirmed by EThcD spectra are annotated and color coded in red as S-ADPr and in blue as R-ADPr sites. ADP-ribosylated peptides with uncertain ADP-ribosylation site localization are shown in black. The black hyperbolic line represents a permutation-based false discovery rate (FDR) of 5% and a minimal fold change of 2. d Normalized abundance of individual Ser- and Arg-ADPr peptides after PARG or ARH3 treatment. Data represent means ± SEM for n = 3 independent demodification experiments

    Techniques Used: In Vitro

    ARH3 regulates basal and hydrogen peroxide-induced serine ADP-ribosylation in vivo. a Venn diagrams of unique ADP-ribosylated peptides of wild type (WT) and ARH3 KO MEF cells under basal and H 2 O 2 -treated conditions. b Unique ADP-ribosylation sites detected by EThcD fragmentation in the different samples. c Gene ontology analysis of the identified ADPr-modified proteins using the PANTHER database. Shown on the left are the P -values and on the right the number of identified and annotated ADP-ribosylated proteins. d Validation of mono-ARH activity of ARH3 on the nuclear protein HMGB1. Recombinant HMGB1 was in vitro ADP-ribosylated using recombinant ARTD1 in the presence of 32 P-labeled NAD + . Equal fractions were left untreated (Input) or were treated with PARG or ARH3. Above: radioactivity exposure, below: Coomassie Blue-stained poly-acrylamide gel. e Motif searches for ADP-ribosylated peptides with a mascot site localization score > 80% in MEF cells using Weblogo. f Energy minimized binding mode of an acetyl-KSG peptide with ADPr-Ser modification. The surface of ARH3 (including the binding-site magnesium ions) is colored according to electrostatic potential (on a scale of −5 to 5 kT/e). The positively charged amino group of the K side chain and the backbone amide groups point toward the region of the surface with negative potential
    Figure Legend Snippet: ARH3 regulates basal and hydrogen peroxide-induced serine ADP-ribosylation in vivo. a Venn diagrams of unique ADP-ribosylated peptides of wild type (WT) and ARH3 KO MEF cells under basal and H 2 O 2 -treated conditions. b Unique ADP-ribosylation sites detected by EThcD fragmentation in the different samples. c Gene ontology analysis of the identified ADPr-modified proteins using the PANTHER database. Shown on the left are the P -values and on the right the number of identified and annotated ADP-ribosylated proteins. d Validation of mono-ARH activity of ARH3 on the nuclear protein HMGB1. Recombinant HMGB1 was in vitro ADP-ribosylated using recombinant ARTD1 in the presence of 32 P-labeled NAD + . Equal fractions were left untreated (Input) or were treated with PARG or ARH3. Above: radioactivity exposure, below: Coomassie Blue-stained poly-acrylamide gel. e Motif searches for ADP-ribosylated peptides with a mascot site localization score > 80% in MEF cells using Weblogo. f Energy minimized binding mode of an acetyl-KSG peptide with ADPr-Ser modification. The surface of ARH3 (including the binding-site magnesium ions) is colored according to electrostatic potential (on a scale of −5 to 5 kT/e). The positively charged amino group of the K side chain and the backbone amide groups point toward the region of the surface with negative potential

    Techniques Used: In Vivo, Modification, Activity Assay, Recombinant, In Vitro, Labeling, Radioactivity, Staining, Acrylamide Gel Assay, Binding Assay

    ARH3 has mono-ARH activity. a Left panel: Recombinant H3 histone tail was in vitro ADP-ribosylated using recombinant ARTD1 in the presence of 32 P-labeled NAD + . Equal fractions were left untreated (Input) or were treated with PARG or ARH3. Above: radioactivity exposure, below: Coomassie Blue-stained poly-acrylamide gel. Right panel: Quantification of a expressed as demodification activity (=reduction of the radioactive signal, normalized to amount of protein). Data represent means ± SEM for n = 3 independent experiments, *** P
    Figure Legend Snippet: ARH3 has mono-ARH activity. a Left panel: Recombinant H3 histone tail was in vitro ADP-ribosylated using recombinant ARTD1 in the presence of 32 P-labeled NAD + . Equal fractions were left untreated (Input) or were treated with PARG or ARH3. Above: radioactivity exposure, below: Coomassie Blue-stained poly-acrylamide gel. Right panel: Quantification of a expressed as demodification activity (=reduction of the radioactive signal, normalized to amount of protein). Data represent means ± SEM for n = 3 independent experiments, *** P

    Techniques Used: Activity Assay, Recombinant, In Vitro, Labeling, Radioactivity, Staining, Acrylamide Gel Assay

    14) Product Images from "Regioselectivity of oxidation by a polysaccharide monooxygenase from Chaetomium thermophilum"

    Article Title: Regioselectivity of oxidation by a polysaccharide monooxygenase from Chaetomium thermophilum

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-018-1156-2

    Mutated CtPMO1 insoluble reaction products oxidized by Br 2 with PASC as substrate using MALDI-TOF–MS. Insoluble reaction products (residual PASC) upon incubation of 0.5% PASC with mutated CtPMO1 Y27A ( a ), H64A ( b ), H157A ( c ) and Y206A ( d ) in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. C1-oxidized oligosaccharides ( m/z + 16), C4-oxidized oligosaccharides ( m/z + 14), and C6-oxidized oligosaccharides ( m/z + 30)
    Figure Legend Snippet: Mutated CtPMO1 insoluble reaction products oxidized by Br 2 with PASC as substrate using MALDI-TOF–MS. Insoluble reaction products (residual PASC) upon incubation of 0.5% PASC with mutated CtPMO1 Y27A ( a ), H64A ( b ), H157A ( c ) and Y206A ( d ) in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. C1-oxidized oligosaccharides ( m/z + 16), C4-oxidized oligosaccharides ( m/z + 14), and C6-oxidized oligosaccharides ( m/z + 30)

    Techniques Used: Mass Spectrometry, Incubation, HAC Assay

    Identification of CtPMO1 reaction products oxidized by Br 2 with celloheptaose as substrate using MALDI-TOF–MS. Reaction products upon incubation of 0.1% celloheptaose with CtPMO1 in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. Reaction products ( a ) and reaction products oxidized by Br 2 ( b ). C1-oxidized oligosaccharides ( m/z + 16), C4- or C6-oxidized oligosaccharides ( m/z − 2), C4- and C1-oxidized oligosaccharides ( m/z + 14), C6- and C1-oxidized oligosaccharides ( m/z + 30), and C1-, C6- and C4-oxidized oligosaccharides ( m/z + 28)
    Figure Legend Snippet: Identification of CtPMO1 reaction products oxidized by Br 2 with celloheptaose as substrate using MALDI-TOF–MS. Reaction products upon incubation of 0.1% celloheptaose with CtPMO1 in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. Reaction products ( a ) and reaction products oxidized by Br 2 ( b ). C1-oxidized oligosaccharides ( m/z + 16), C4- or C6-oxidized oligosaccharides ( m/z − 2), C4- and C1-oxidized oligosaccharides ( m/z + 14), C6- and C1-oxidized oligosaccharides ( m/z + 30), and C1-, C6- and C4-oxidized oligosaccharides ( m/z + 28)

    Techniques Used: Mass Spectrometry, Incubation, HAC Assay

    Identification of the mutated CtPMO1 soluble reaction products oxidized by Br 2 with PASC as substrate using MALDI-TOF–MS. Soluble reaction products upon incubation of 0.5% PASC with the mutated CtPMO1 enzymes Y27A ( a ), H64A ( b ), H157A ( c ) and Y206A ( d ) in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. C1-oxidized oligosaccharides ( m/z + 16), C4-oxidized oligosaccharides ( m/z + 14), C6-oxidized oligosaccharides ( m/z + 30), and C6- and C4-oxidized oligosaccharides ( m/z + 28)
    Figure Legend Snippet: Identification of the mutated CtPMO1 soluble reaction products oxidized by Br 2 with PASC as substrate using MALDI-TOF–MS. Soluble reaction products upon incubation of 0.5% PASC with the mutated CtPMO1 enzymes Y27A ( a ), H64A ( b ), H157A ( c ) and Y206A ( d ) in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. C1-oxidized oligosaccharides ( m/z + 16), C4-oxidized oligosaccharides ( m/z + 14), C6-oxidized oligosaccharides ( m/z + 30), and C6- and C4-oxidized oligosaccharides ( m/z + 28)

    Techniques Used: Mass Spectrometry, Incubation, HAC Assay

    Identification of the mutated CtPMO1 soluble reaction products with PASC as substrate using TLC. Soluble reaction products upon incubation of 0.5% PASC with the mutated CtPMO1 enzymes (Y27A, H64A, H157A and Y206A) in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. M, standard cellulo-oligosaccharides (G1–G7). CK, samples upon incubation of 0.5% PASC in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h with inactive CtPMO1 treated at 100 °C for 30 min
    Figure Legend Snippet: Identification of the mutated CtPMO1 soluble reaction products with PASC as substrate using TLC. Soluble reaction products upon incubation of 0.5% PASC with the mutated CtPMO1 enzymes (Y27A, H64A, H157A and Y206A) in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. M, standard cellulo-oligosaccharides (G1–G7). CK, samples upon incubation of 0.5% PASC in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h with inactive CtPMO1 treated at 100 °C for 30 min

    Techniques Used: Thin Layer Chromatography, Incubation, HAC Assay

    Identification of the mutated CtPMO1 soluble reaction products with PASC as substrate using MALDI-TOF–MS. Soluble reaction products upon incubation of 0.5% PASC with the mutated CtPMO1 enzymes Y27A ( a ), H64A ( b ), H157A ( c ) and Y206A ( d ) in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. C1-oxidized oligosaccharides (aldonic acid, m/z + 16) and C4- or C6-oxidized oligosaccharides (C4-ketoaldose or C6-hexodialdose, m/z − 2)
    Figure Legend Snippet: Identification of the mutated CtPMO1 soluble reaction products with PASC as substrate using MALDI-TOF–MS. Soluble reaction products upon incubation of 0.5% PASC with the mutated CtPMO1 enzymes Y27A ( a ), H64A ( b ), H157A ( c ) and Y206A ( d ) in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. C1-oxidized oligosaccharides (aldonic acid, m/z + 16) and C4- or C6-oxidized oligosaccharides (C4-ketoaldose or C6-hexodialdose, m/z − 2)

    Techniques Used: Mass Spectrometry, Incubation, HAC Assay

    Analysis of CtPMO1 soluble reaction products with PASC as substrate using TLC. Soluble reaction products upon incubation of 0.5% PASC with CtPMO1 in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 0, 12, 24, 36, and 48 h. M, standard cellulo-oligosaccharides (G1–G7)
    Figure Legend Snippet: Analysis of CtPMO1 soluble reaction products with PASC as substrate using TLC. Soluble reaction products upon incubation of 0.5% PASC with CtPMO1 in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 0, 12, 24, 36, and 48 h. M, standard cellulo-oligosaccharides (G1–G7)

    Techniques Used: Thin Layer Chromatography, Incubation, HAC Assay

    Identification of CtPMO1 soluble reaction products with PASC as substrate using MALDI-TOF–MS. Soluble reaction products upon incubation of 0.5% PASC with CtPMO1 in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h
    Figure Legend Snippet: Identification of CtPMO1 soluble reaction products with PASC as substrate using MALDI-TOF–MS. Soluble reaction products upon incubation of 0.5% PASC with CtPMO1 in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h

    Techniques Used: Mass Spectrometry, Incubation, HAC Assay

    CtPMO1 insoluble reaction products hydrolyzed by endo-1,4-beta-glucanase followed by Br 2 oxidation with PASC as substrate. Insoluble reaction products (residual PASC) upon incubation of 0.5% PASC with CtPMO1 in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. CtPMO1 insoluble reaction products hydrolyzed by endo-1,4-beta-glucanase using TLC analysis ( a ) and MALDI-TOF–MS ( b ). CtPMO1 insoluble reaction products hydrolyzed by endo-1,4-beta-glucanase followed by Br 2 oxidation for 30 min ( c ) and for 60 min ( d ) using MALDI-TOF–MS analysis. C1-oxidized oligosaccharides ( m/z + 16), C4- or C6-oxidized oligosaccharides ( m/z − 2), lactones ( m/z -4) of C1- ( m/z + 16) and C4- or C6-oxidized oligosaccharides ( m/z − 2), C4- and C1-oxidized oligosaccharides ( m/z + 14), C6- and C1-oxidized oligosaccharides ( m/z + 30), and C6-, C1- and C4-oxidized oligosaccharides ( m/z + 28). M, standard cellulo-oligosaccharides (G1–G7). CK, samples upon incubation of insoluble reaction products (residual PASC) in 10 mM HAc-NH 4 Ac (pH 5.0) at 50 °C for 10 min with inactive endo-1,4-beta-glucanase treated at 100 °C for 30 min
    Figure Legend Snippet: CtPMO1 insoluble reaction products hydrolyzed by endo-1,4-beta-glucanase followed by Br 2 oxidation with PASC as substrate. Insoluble reaction products (residual PASC) upon incubation of 0.5% PASC with CtPMO1 in 10 mM HAc-NH 4 Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. CtPMO1 insoluble reaction products hydrolyzed by endo-1,4-beta-glucanase using TLC analysis ( a ) and MALDI-TOF–MS ( b ). CtPMO1 insoluble reaction products hydrolyzed by endo-1,4-beta-glucanase followed by Br 2 oxidation for 30 min ( c ) and for 60 min ( d ) using MALDI-TOF–MS analysis. C1-oxidized oligosaccharides ( m/z + 16), C4- or C6-oxidized oligosaccharides ( m/z − 2), lactones ( m/z -4) of C1- ( m/z + 16) and C4- or C6-oxidized oligosaccharides ( m/z − 2), C4- and C1-oxidized oligosaccharides ( m/z + 14), C6- and C1-oxidized oligosaccharides ( m/z + 30), and C6-, C1- and C4-oxidized oligosaccharides ( m/z + 28). M, standard cellulo-oligosaccharides (G1–G7). CK, samples upon incubation of insoluble reaction products (residual PASC) in 10 mM HAc-NH 4 Ac (pH 5.0) at 50 °C for 10 min with inactive endo-1,4-beta-glucanase treated at 100 °C for 30 min

    Techniques Used: Incubation, HAC Assay, Thin Layer Chromatography, Mass Spectrometry

    Identification of CtPMO1 soluble reaction products oxidized by Br 2 with PASC as substrate using MALDI-TOF–MS. Reaction pathway for oxidation of cellulose by CtPMO1 followed Br 2 oxidation ( a ) and reaction products oxidized by Br 2 ( b ). C1-oxidized oligosaccharides ( m/z + 16), C4- and C1-oxidized oligosaccharides ( m/z + 14), C1- and C6-oxidized oligosaccharides ( m/z + 30), and C1-, C6- and C4-oxidized oligosaccharides ( m/z + 28)
    Figure Legend Snippet: Identification of CtPMO1 soluble reaction products oxidized by Br 2 with PASC as substrate using MALDI-TOF–MS. Reaction pathway for oxidation of cellulose by CtPMO1 followed Br 2 oxidation ( a ) and reaction products oxidized by Br 2 ( b ). C1-oxidized oligosaccharides ( m/z + 16), C4- and C1-oxidized oligosaccharides ( m/z + 14), C1- and C6-oxidized oligosaccharides ( m/z + 30), and C1-, C6- and C4-oxidized oligosaccharides ( m/z + 28)

    Techniques Used: Mass Spectrometry

    15) Product Images from "Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival"

    Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

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

    doi: 10.1073/pnas.1601053113

    ( A ) Protein expression profiling: Scatter plots of normalized “protein groups” SILAC ratios (log 2 ) versus intensity (log 10 ) for 2, 5, 10, and 20 min after BCR stimulation. Outlier proteins are marked red, and the respective gene names are shown. ( B ) Pearson’s correlation analysis of normalized SILAC ratios (log 2 ) of all p-sites quantified in four biological replicates (indicated by 1, 2, 3, and 4) from the global phosphoproteome experiment of DG75 cells. Pearson’s correlation coefficient is indicated in blue in the upper left corner of each scatter plot. The pink box highlights the correlation of the biological replicates of 2 min, blue 5 min, yellow 10 min, and green 20 min BCR stimulation. ( C ) Pearson’s correlation analysis of normalized SILAC ratios (log 2 ) of biological replicates (indicated by 1 and 2) of all p-sites derived from the pYome experiment. Pearson’s correlation coefficient is shown in blue in the upper left corner of each scatter plot. The pink box highlights the correlation of the biological replicates of 2 min, blue 5 min, yellow 10 min, and green 20 min BCR stimulation.
    Figure Legend Snippet: ( A ) Protein expression profiling: Scatter plots of normalized “protein groups” SILAC ratios (log 2 ) versus intensity (log 10 ) for 2, 5, 10, and 20 min after BCR stimulation. Outlier proteins are marked red, and the respective gene names are shown. ( B ) Pearson’s correlation analysis of normalized SILAC ratios (log 2 ) of all p-sites quantified in four biological replicates (indicated by 1, 2, 3, and 4) from the global phosphoproteome experiment of DG75 cells. Pearson’s correlation coefficient is indicated in blue in the upper left corner of each scatter plot. The pink box highlights the correlation of the biological replicates of 2 min, blue 5 min, yellow 10 min, and green 20 min BCR stimulation. ( C ) Pearson’s correlation analysis of normalized SILAC ratios (log 2 ) of biological replicates (indicated by 1 and 2) of all p-sites derived from the pYome experiment. Pearson’s correlation coefficient is shown in blue in the upper left corner of each scatter plot. The pink box highlights the correlation of the biological replicates of 2 min, blue 5 min, yellow 10 min, and green 20 min BCR stimulation.

    Techniques Used: Expressing, Derivative Assay

    Activated BCR signaling in BL cells. ( A ) Numbers of quantified and regulated p-sites in the GPome and the pYome of DG75 cells. Outer circles represent the number of quantified class I p-sites at all stimulation durations. Inner circles represent the relative abundance of regulated class I p-sites. ( B ) Unsupervised clustering analysis of all regulated p-sites quantified at all BCR stimulation durations in DG75 cells. Line graphs illustrate the phosphorylation dynamics upon BCR stimulation for each identified cluster for the indicated stimulation time course ( y axis, z-score of the log 2 SILAC ratios; x axis, minutes). ( C – F ) Scatter plots of fold-change of p-sites on tyrosines ( C and E ) and serines/threonines ( D and F ) in Daudi ( y axis) versus DG75 ( x axis) cells as determined by quantitative MS upon 2-min ( C and D ) and 10-min ( E and F ) BCR stimulation. Selected phosphorylated proteins are shown. Colored dots indicate p-sites that are significantly regulated in both cell lines (red) or just in one cell line (blue). The Spearman’s correlation coefficient (ρ) is indicated.
    Figure Legend Snippet: Activated BCR signaling in BL cells. ( A ) Numbers of quantified and regulated p-sites in the GPome and the pYome of DG75 cells. Outer circles represent the number of quantified class I p-sites at all stimulation durations. Inner circles represent the relative abundance of regulated class I p-sites. ( B ) Unsupervised clustering analysis of all regulated p-sites quantified at all BCR stimulation durations in DG75 cells. Line graphs illustrate the phosphorylation dynamics upon BCR stimulation for each identified cluster for the indicated stimulation time course ( y axis, z-score of the log 2 SILAC ratios; x axis, minutes). ( C – F ) Scatter plots of fold-change of p-sites on tyrosines ( C and E ) and serines/threonines ( D and F ) in Daudi ( y axis) versus DG75 ( x axis) cells as determined by quantitative MS upon 2-min ( C and D ) and 10-min ( E and F ) BCR stimulation. Selected phosphorylated proteins are shown. Colored dots indicate p-sites that are significantly regulated in both cell lines (red) or just in one cell line (blue). The Spearman’s correlation coefficient (ρ) is indicated.

    Techniques Used: Mass Spectrometry

    16) Product Images from "Selected imprinting of INS in the marsupial"

    Article Title: Selected imprinting of INS in the marsupial

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-5-14

    INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.
    Figure Legend Snippet: INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.

    Techniques Used: Sequencing, Expressing, Produced

    Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.
    Figure Legend Snippet: Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.

    Techniques Used: Methylation, Rapid Amplification of cDNA Ends, Derivative Assay, Amplification

    17) Product Images from "Translating Divergent Environmental Stresses into a Common Proteome Response through the Histidine Kinase 33 (Hik33) in a Model Cyanobacterium *"

    Article Title: Translating Divergent Environmental Stresses into a Common Proteome Response through the Histidine Kinase 33 (Hik33) in a Model Cyanobacterium *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M116.068080

    Rationale and experimental design. A , Confirmation of Δ hik33 growth phenotype under photoautotrophic condition. The deletion of hik33 ), which also contains the photo of the cell culture. B , The WCLs of the WT and Δ hik33 strains of Synechocystis were separated into the membrane (Mem) and the soluble (Sol) fractions. Proteins extracted from either fraction and the WCL were subsequently separated by SDS-PAGE and visualized with Coomassie blue staining. C , Schematic representation of the workflow for the quantitative analysis of the Δ hik33 proteome. Total proteins were extracted from the WCLs of three biological replicates of the WT and Δ hik33 and digested with trypsin. The tryptic peptides were labeled with 6-plex TMT reagents in the order as indicated. The labeled peptides were mixed together with an equal molar ratio, and separated into 15 fractions with RP-HPLC. The peptides in each fraction were quantitatively analyzed by LC-MS/MS using a LTQ-Orbitrap-Elite mass spectrometer.
    Figure Legend Snippet: Rationale and experimental design. A , Confirmation of Δ hik33 growth phenotype under photoautotrophic condition. The deletion of hik33 ), which also contains the photo of the cell culture. B , The WCLs of the WT and Δ hik33 strains of Synechocystis were separated into the membrane (Mem) and the soluble (Sol) fractions. Proteins extracted from either fraction and the WCL were subsequently separated by SDS-PAGE and visualized with Coomassie blue staining. C , Schematic representation of the workflow for the quantitative analysis of the Δ hik33 proteome. Total proteins were extracted from the WCLs of three biological replicates of the WT and Δ hik33 and digested with trypsin. The tryptic peptides were labeled with 6-plex TMT reagents in the order as indicated. The labeled peptides were mixed together with an equal molar ratio, and separated into 15 fractions with RP-HPLC. The peptides in each fraction were quantitatively analyzed by LC-MS/MS using a LTQ-Orbitrap-Elite mass spectrometer.

    Techniques Used: Cell Culture, SDS Page, Staining, Labeling, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    18) Product Images from "Low Molecular Mass Myocardial Hyaluronan in Human Hypertrophic Cardiomyopathy"

    Article Title: Low Molecular Mass Myocardial Hyaluronan in Human Hypertrophic Cardiomyopathy

    Journal: Cells

    doi: 10.3390/cells8020097

    Factor analysis for correlation of gene expression levels. HAS1-3 , HYAL1-2 , CEMIP , CD44 , VCAN , and TSG6 in non-failing left wall and septum (n = 9) ( a ) and HCM myectomies (n = 5) ( b ). ( a ) In left wall and septum there are two clusters where HAS1 , CEMIP , CD44 , VCAN , and TSG6 form one cluster, and HAS2 , HAS3, and HYAL2 form another. ( b ) In basal septal myectomies from HCM patients the expression levels of CEMIP , CD44, and VCAN formed a new correlation cluster with HAS3 . HAS2 , HYAL1 , and TSG6 formed another cluster. The levels of HAS1 and HYAL2 no longer correlated with any of the other genes investigated. Factor analysis was performed with the principal components method to analyze the correlation matrix and two factors were extracted.
    Figure Legend Snippet: Factor analysis for correlation of gene expression levels. HAS1-3 , HYAL1-2 , CEMIP , CD44 , VCAN , and TSG6 in non-failing left wall and septum (n = 9) ( a ) and HCM myectomies (n = 5) ( b ). ( a ) In left wall and septum there are two clusters where HAS1 , CEMIP , CD44 , VCAN , and TSG6 form one cluster, and HAS2 , HAS3, and HYAL2 form another. ( b ) In basal septal myectomies from HCM patients the expression levels of CEMIP , CD44, and VCAN formed a new correlation cluster with HAS3 . HAS2 , HYAL1 , and TSG6 formed another cluster. The levels of HAS1 and HYAL2 no longer correlated with any of the other genes investigated. Factor analysis was performed with the principal components method to analyze the correlation matrix and two factors were extracted.

    Techniques Used: Expressing

    19) Product Images from "Bi-allelic Mutations in Phe-tRNA Synthetase Associated with a Multi-system Pulmonary Disease Support Non-translational Function"

    Article Title: Bi-allelic Mutations in Phe-tRNA Synthetase Associated with a Multi-system Pulmonary Disease Support Non-translational Function

    Journal: American Journal of Human Genetics

    doi: 10.1016/j.ajhg.2018.06.006

    The c848+1G > A Intronic Mutation Resulted in Aberrant Splicing of FARSB (A) The mutation of the universal splice site motif is expected to affect the splicing of nearby exons in the FARSB gene, such as skipping of the adjacent exon 9. (B) A splice variant that deleted exon 9 of the FARSB _FL transcript, designated as FARSB -ΔE9, was detected in the proband but not in the mother. (C) Quantitative real-time PCR analysis of mRNA expressions of FARSB _E9-11 (using primers targeting exons 9 and 11 of FARSB , thus primarily detecting the full-length transcript), and FARSB -ΔE9 (using primers specifically amplifying the ΔE9 variant) in primary fibroblasts. Gene expression of FARSB _E9-11 and -ΔE9 in the fibroblasts of the proband, both parents, and three control subjects were calculated based on Ct values normalized to house-keeping genes RPL9 and RPS11 . The fold changes were thus calculated by relative to the FARSB _E9-11 level of CTL-17M. Data were presented as mean ± SEM. The ΔE9 RNA was detected only in the proband and father cells but not in the mother and control subjects (U.D. denotes no detectable amplification within 45 qPCR cycles).
    Figure Legend Snippet: The c848+1G > A Intronic Mutation Resulted in Aberrant Splicing of FARSB (A) The mutation of the universal splice site motif is expected to affect the splicing of nearby exons in the FARSB gene, such as skipping of the adjacent exon 9. (B) A splice variant that deleted exon 9 of the FARSB _FL transcript, designated as FARSB -ΔE9, was detected in the proband but not in the mother. (C) Quantitative real-time PCR analysis of mRNA expressions of FARSB _E9-11 (using primers targeting exons 9 and 11 of FARSB , thus primarily detecting the full-length transcript), and FARSB -ΔE9 (using primers specifically amplifying the ΔE9 variant) in primary fibroblasts. Gene expression of FARSB _E9-11 and -ΔE9 in the fibroblasts of the proband, both parents, and three control subjects were calculated based on Ct values normalized to house-keeping genes RPL9 and RPS11 . The fold changes were thus calculated by relative to the FARSB _E9-11 level of CTL-17M. Data were presented as mean ± SEM. The ΔE9 RNA was detected only in the proband and father cells but not in the mother and control subjects (U.D. denotes no detectable amplification within 45 qPCR cycles).

    Techniques Used: Mutagenesis, Variant Assay, Real-time Polymerase Chain Reaction, Expressing, CTL Assay, Amplification

    20) Product Images from "Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status"

    Article Title: Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    Journal: Nature Communications

    doi: 10.1038/ncomms12645

    Deep single-run proteomics of cell lines and human ovarian cancer tissue. ( a ) Summary of the shotgun proteomics workflow for OvCa cellular models and high-grade serous ovarian cancer (HGSOC) tissues. Following lysis, protein purification, and tryptic digest, peptides were separated by ultra-high performance liquid chromatography and measured in single runs using a quadrupole Orbitrap mass spectrometer. Label-free proteome quantification was performed using the MaxQuant software environment. ( b ) Workflow reproducibility for cell lines ( n =30), HGSOC tissues ( n =8) and primary FTEC cells ( n =3). Pearson correlations ( r ) were calculated for biological replicates of cell lines, primary FTEC isolates from different healthy donors, and ovarian tumour tissues from both ovaries from a woman with HGSOC. ( c ) Average number of quantified proteins from each sample type. Error bars represent standard deviations. ( d ) Number of proteins common to FTECs, HGSOC tumours and cell lines. FTEC, fallopian tube epithelial cell; LC-MS/MS, liquid chromatography tandem mass spectrometry; m/z , mass-to-charge ratio; uHPLC, ultra-high performance liquid chromatography.
    Figure Legend Snippet: Deep single-run proteomics of cell lines and human ovarian cancer tissue. ( a ) Summary of the shotgun proteomics workflow for OvCa cellular models and high-grade serous ovarian cancer (HGSOC) tissues. Following lysis, protein purification, and tryptic digest, peptides were separated by ultra-high performance liquid chromatography and measured in single runs using a quadrupole Orbitrap mass spectrometer. Label-free proteome quantification was performed using the MaxQuant software environment. ( b ) Workflow reproducibility for cell lines ( n =30), HGSOC tissues ( n =8) and primary FTEC cells ( n =3). Pearson correlations ( r ) were calculated for biological replicates of cell lines, primary FTEC isolates from different healthy donors, and ovarian tumour tissues from both ovaries from a woman with HGSOC. ( c ) Average number of quantified proteins from each sample type. Error bars represent standard deviations. ( d ) Number of proteins common to FTECs, HGSOC tumours and cell lines. FTEC, fallopian tube epithelial cell; LC-MS/MS, liquid chromatography tandem mass spectrometry; m/z , mass-to-charge ratio; uHPLC, ultra-high performance liquid chromatography.

    Techniques Used: Lysis, Protein Purification, High Performance Liquid Chromatography, Mass Spectrometry, Software, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    21) Product Images from "Adsorption of Azo Dye Methyl Orange from Aqueous Solutions Using Alkali-Activated Polypyrrole-Based Graphene Oxide"

    Article Title: Adsorption of Azo Dye Methyl Orange from Aqueous Solutions Using Alkali-Activated Polypyrrole-Based Graphene Oxide

    Journal: Molecules

    doi: 10.3390/molecules24203685

    Fourier transform infrared (FTIR) spectra of Polypyrrole (PPy), KOH-activated polypyrrole-based adsorbent (PACK), PACK–MO, and methyl orange (MO).
    Figure Legend Snippet: Fourier transform infrared (FTIR) spectra of Polypyrrole (PPy), KOH-activated polypyrrole-based adsorbent (PACK), PACK–MO, and methyl orange (MO).

    Techniques Used:

    22) Product Images from "Selected imprinting of INS in the marsupial"

    Article Title: Selected imprinting of INS in the marsupial

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-5-14

    INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.
    Figure Legend Snippet: INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.

    Techniques Used: Sequencing, Expressing, Produced

    Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.
    Figure Legend Snippet: Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.

    Techniques Used: Methylation, Rapid Amplification of cDNA Ends, Derivative Assay, Amplification

    23) Product Images from "A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans"

    Article Title: A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-018-1074-3

    MALDI-TOF spectra of feruloylated and/or acetylated AX oligomers before ( a ) and after incubation with FjoAcXE ( b ]; P pentose, A arabinose, G galactose, Ac acetyl, FA feruloyl. The m/z values represent sodium adducts
    Figure Legend Snippet: MALDI-TOF spectra of feruloylated and/or acetylated AX oligomers before ( a ) and after incubation with FjoAcXE ( b ]; P pentose, A arabinose, G galactose, Ac acetyl, FA feruloyl. The m/z values represent sodium adducts

    Techniques Used: Incubation

    1 H-NMR spectrum of 1% (w/v) hot water extracted glucuronoxylan following treatment with FjoAcXE. The solid arrow indicates the unique ability of FjoAcXE to deacetylate 3- O -acetylated Xyl p 2- O -substituted with MeGlc p ]. Reactions were performed in 50 mM HEPES (pH 7.0) for 20 h at 30 °C; reactions were then lyophilized and suspended in 0.3 mL HDO. *100% represents 0.29 mg/mL release of (Me)Glc p ]
    Figure Legend Snippet: 1 H-NMR spectrum of 1% (w/v) hot water extracted glucuronoxylan following treatment with FjoAcXE. The solid arrow indicates the unique ability of FjoAcXE to deacetylate 3- O -acetylated Xyl p 2- O -substituted with MeGlc p ]. Reactions were performed in 50 mM HEPES (pH 7.0) for 20 h at 30 °C; reactions were then lyophilized and suspended in 0.3 mL HDO. *100% represents 0.29 mg/mL release of (Me)Glc p ]

    Techniques Used: Nuclear Magnetic Resonance

    PUL20 Flavobacterium johnsoniae showing possible functions of predicted carbohydrate-active enzymes (CAZymes). Proteins annotated as having unknown function (unk) are shown in gray. The red arrow indicates the gene encoding FjoAcXE (Fjoh_3879). Predicted CAZymes include glycoside hydrolase (GH) and carbohydrate esterase (CE) families that comprise xylan-active enzymes. A vertical slash between two modules represents multidomain CAZymes, and arrows indicate whether the gene is found on the sense (right pointing triangle) or antisense (left pointing triangle) strand
    Figure Legend Snippet: PUL20 Flavobacterium johnsoniae showing possible functions of predicted carbohydrate-active enzymes (CAZymes). Proteins annotated as having unknown function (unk) are shown in gray. The red arrow indicates the gene encoding FjoAcXE (Fjoh_3879). Predicted CAZymes include glycoside hydrolase (GH) and carbohydrate esterase (CE) families that comprise xylan-active enzymes. A vertical slash between two modules represents multidomain CAZymes, and arrows indicate whether the gene is found on the sense (right pointing triangle) or antisense (left pointing triangle) strand

    Techniques Used:

    General biochemical properties of FjoAcXE. For optimum pH ( a ) and pH stability ( b ), the samples were tested using 100 mM Tris, 50 mM MES, 50 mM acetic acid, and 50 mM sodium acetate trihydrate universal buffer, with pH range of 3.5–9.5. Temperature stability ( c ), effect of metal ions ( d ), and effect of selected solvents and reactants ( e ), were measured using 0.5 mM 4-MUA in 50 mM HEPES (pH 8.0) where reactions proceeded for 20 min at 30 °C. Absorbance at 354 nm was measured and the reaction mixture without protein was used as a blank. n = 3; error bars correspond to standard deviation
    Figure Legend Snippet: General biochemical properties of FjoAcXE. For optimum pH ( a ) and pH stability ( b ), the samples were tested using 100 mM Tris, 50 mM MES, 50 mM acetic acid, and 50 mM sodium acetate trihydrate universal buffer, with pH range of 3.5–9.5. Temperature stability ( c ), effect of metal ions ( d ), and effect of selected solvents and reactants ( e ), were measured using 0.5 mM 4-MUA in 50 mM HEPES (pH 8.0) where reactions proceeded for 20 min at 30 °C. Absorbance at 354 nm was measured and the reaction mixture without protein was used as a blank. n = 3; error bars correspond to standard deviation

    Techniques Used: Standard Deviation

    24) Product Images from "Antiprotozoal Activity of Buxus sempervirens and Activity-Guided Isolation of O-tigloylcyclovirobuxeine-B as the Main Constituent Active against Plasmodium falciparum ‡"

    Article Title: Antiprotozoal Activity of Buxus sempervirens and Activity-Guided Isolation of O-tigloylcyclovirobuxeine-B as the Main Constituent Active against Plasmodium falciparum ‡

    Journal: Molecules

    doi: 10.3390/molecules19056184

    UHPLC/+ESI-QTOF-MSMS analysis of O -tigloylcyclovirobuxeine-B ( 1 ) (top) and of an aqueous decoction as used in ethnomedicine (bottom). Blue: Base peak chromatogram ( m/z 100–1000); Red: EIC m/z 497 [M+H] + ; Magenta: EIC m/z 249 [M+2H] 2+ .
    Figure Legend Snippet: UHPLC/+ESI-QTOF-MSMS analysis of O -tigloylcyclovirobuxeine-B ( 1 ) (top) and of an aqueous decoction as used in ethnomedicine (bottom). Blue: Base peak chromatogram ( m/z 100–1000); Red: EIC m/z 497 [M+H] + ; Magenta: EIC m/z 249 [M+2H] 2+ .

    Techniques Used:

    UHPLC/+ESI-QTOF-MS chromatograms of ( A ) the crude extract, ( B ) the alkaloid fraction ALK, ( C ) the non-polar neutral fraction APO and ( D ) of subfraction E 59. Blue: Base peak chromatogram of m/z 100–1000, Red: Extracted ion chromatogram of m/z 497 [M+H] + ; Magenta: extracted ion chromatogram of m/z 249 [M+2H] 2+ .
    Figure Legend Snippet: UHPLC/+ESI-QTOF-MS chromatograms of ( A ) the crude extract, ( B ) the alkaloid fraction ALK, ( C ) the non-polar neutral fraction APO and ( D ) of subfraction E 59. Blue: Base peak chromatogram of m/z 100–1000, Red: Extracted ion chromatogram of m/z 497 [M+H] + ; Magenta: extracted ion chromatogram of m/z 249 [M+2H] 2+ .

    Techniques Used: Mass Spectrometry

    25) Product Images from "Delta-Opioid Receptor Analgesia Is Independent of Microglial Activation in a Rat Model of Neuropathic Pain"

    Article Title: Delta-Opioid Receptor Analgesia Is Independent of Microglial Activation in a Rat Model of Neuropathic Pain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104420

    MOR , DOR , KOR mRNAs in spinal cord and DRGs in vehicle- and minocycline-treated CCI-exposed rats. Minocycline (MC; 30 mg/kg; i.p.) was administered intraperitoneally pre-emptively 16 h and 1 h before CCI, and then repeatedly twice daily for 7 days. On the seventh day, spinal cords (L4–L6) and DRG were collected for the qRT-PCR analysis of MOR (A, B), KOR (C, D) and DOR (E, F) gene expression. The data are presented as the means ± SEM and represent the normalised averages derived from the threshold qRT-PCR cycles from four to eight samples for each group. Intergroup differences were analysed using ANOVAs followed by Bonferroni's multiple comparison tests. * P
    Figure Legend Snippet: MOR , DOR , KOR mRNAs in spinal cord and DRGs in vehicle- and minocycline-treated CCI-exposed rats. Minocycline (MC; 30 mg/kg; i.p.) was administered intraperitoneally pre-emptively 16 h and 1 h before CCI, and then repeatedly twice daily for 7 days. On the seventh day, spinal cords (L4–L6) and DRG were collected for the qRT-PCR analysis of MOR (A, B), KOR (C, D) and DOR (E, F) gene expression. The data are presented as the means ± SEM and represent the normalised averages derived from the threshold qRT-PCR cycles from four to eight samples for each group. Intergroup differences were analysed using ANOVAs followed by Bonferroni's multiple comparison tests. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay

    26) Product Images from "A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation"

    Article Title: A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1259

    pHis levels in PpsA are sensitive to nitrogen availability and are regulated by α-KG a) Western blotting of NCM 3722 cells grown on minimal media containing glucose or glycerol as the carbon source and arginine as the nitrogen source. α-pHis signals were sensitive to hydroxylamine (HA) treatment of the lysates and nitrogen upshift in the growth media (NH 4 Cl). As a loading control, the membranes were imaged with colloidal gold stain ( Supplementary Fig. 21 ). See Supplementary Fig. 22 for full Western blot. b) MS/MS of an endogenous PpsA tryptic pHis peptide identified from fractionated glucose-fed E. coli lysate ( Supplementary Fig. 16 ). The gel band at 85 kDa was analyzed by high-resolution nano-UPLC-MS after trypsin digestion. The spectrum indicates pHis at the canonical His421 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (CAM = S-carboxyamidomethyl) (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). c) Model for regulation of PpsA catalytic cycle. Intracellular levels of α-KG can be significantly increased by nitrogen limitation. Inhibition of the PpsA dephosphorylation by the increased α-KG will lead to the buildup of phosphorylated enzyme. d) Dephosphorylation assay of autophosphorylated PpsA. The dephosphorylation was inhibited by α-KG, but not by glutamate (n = 3, mean ± s.d.).
    Figure Legend Snippet: pHis levels in PpsA are sensitive to nitrogen availability and are regulated by α-KG a) Western blotting of NCM 3722 cells grown on minimal media containing glucose or glycerol as the carbon source and arginine as the nitrogen source. α-pHis signals were sensitive to hydroxylamine (HA) treatment of the lysates and nitrogen upshift in the growth media (NH 4 Cl). As a loading control, the membranes were imaged with colloidal gold stain ( Supplementary Fig. 21 ). See Supplementary Fig. 22 for full Western blot. b) MS/MS of an endogenous PpsA tryptic pHis peptide identified from fractionated glucose-fed E. coli lysate ( Supplementary Fig. 16 ). The gel band at 85 kDa was analyzed by high-resolution nano-UPLC-MS after trypsin digestion. The spectrum indicates pHis at the canonical His421 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (CAM = S-carboxyamidomethyl) (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). c) Model for regulation of PpsA catalytic cycle. Intracellular levels of α-KG can be significantly increased by nitrogen limitation. Inhibition of the PpsA dephosphorylation by the increased α-KG will lead to the buildup of phosphorylated enzyme. d) Dephosphorylation assay of autophosphorylated PpsA. The dephosphorylation was inhibited by α-KG, but not by glutamate (n = 3, mean ± s.d.).

    Techniques Used: Western Blot, Staining, Mass Spectrometry, Sequencing, Chick Chorioallantoic Membrane Assay, Mass Measurement, Inhibition, De-Phosphorylation Assay

    Analysis of histidine phosphorylation on PtsI a) Western blots of E. coli lysates expressing His 6 -tagged PtsI. Left: α-pHis blot of crude lysates and those treated with hydroxylamine (HA) or phosphohistidine phosphatase (PH). Right: Crude lysates were purified over Ni-NTA beads and the indicated fractions probed with the α-pHis antibody. As loading controls, the membranes were stripped and re-blotted with an α-His-tag antibody. See Supplementary Fig. 20 for full Western blots. b) Overexpressed PtsI was digested with trypsin and analyzed by high-resolution nano-UPLC-MS. Shown is the MS/MS spectrum from the tryptic peptide ion bearing pHis at the canonical His-189 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). For comparison, the MS/MS spectrum of a synthetic version of the pHis-bearing peptide is shown in mirror image below. c) A dot blot assay was developed to measure the kinetics of autophosphorylation of PtsI by PEP. A plot of the reaction velocity as a function of PEP concentration was used to determine an apparent K m value of 135 ± 30 μM (n = 3, mean ± s.d.).
    Figure Legend Snippet: Analysis of histidine phosphorylation on PtsI a) Western blots of E. coli lysates expressing His 6 -tagged PtsI. Left: α-pHis blot of crude lysates and those treated with hydroxylamine (HA) or phosphohistidine phosphatase (PH). Right: Crude lysates were purified over Ni-NTA beads and the indicated fractions probed with the α-pHis antibody. As loading controls, the membranes were stripped and re-blotted with an α-His-tag antibody. See Supplementary Fig. 20 for full Western blots. b) Overexpressed PtsI was digested with trypsin and analyzed by high-resolution nano-UPLC-MS. Shown is the MS/MS spectrum from the tryptic peptide ion bearing pHis at the canonical His-189 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). For comparison, the MS/MS spectrum of a synthetic version of the pHis-bearing peptide is shown in mirror image below. c) A dot blot assay was developed to measure the kinetics of autophosphorylation of PtsI by PEP. A plot of the reaction velocity as a function of PEP concentration was used to determine an apparent K m value of 135 ± 30 μM (n = 3, mean ± s.d.).

    Techniques Used: Western Blot, Expressing, Purification, Mass Spectrometry, Sequencing, Mass Measurement, Dot Blot, Concentration Assay

    27) Product Images from "Fabrication of genistein-loaded biodegradable TPGS-b-PCL nanoparticles for improved therapeutic effects in cervical cancer cells"

    Article Title: Fabrication of genistein-loaded biodegradable TPGS-b-PCL nanoparticles for improved therapeutic effects in cervical cancer cells

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S78988

    FTIR spectra of TPGS and TPGS- b -PCL copolymer. Abbreviations: FTIR, Fourier transform infrared; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.
    Figure Legend Snippet: FTIR spectra of TPGS and TPGS- b -PCL copolymer. Abbreviations: FTIR, Fourier transform infrared; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.

    Techniques Used:

    Schematic description of the synthesis of TPGS- b -PCL diblock copolymer. Abbreviations: ε-CL, ε-caprolactone; h, hours; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.
    Figure Legend Snippet: Schematic description of the synthesis of TPGS- b -PCL diblock copolymer. Abbreviations: ε-CL, ε-caprolactone; h, hours; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.

    Techniques Used:

    DSC thermograms of the pure genistein, blank PCL NPs, blank TPGS- b -PCL NPs, and genistein-loaded PCL NPs, and TPGS- b -PCL NPs. Abbreviations: DSC, differential scanning calorimetry; ENDO, endotherm; NPs, nanoparticles; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.
    Figure Legend Snippet: DSC thermograms of the pure genistein, blank PCL NPs, blank TPGS- b -PCL NPs, and genistein-loaded PCL NPs, and TPGS- b -PCL NPs. Abbreviations: DSC, differential scanning calorimetry; ENDO, endotherm; NPs, nanoparticles; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.

    Techniques Used:

    Cellular uptake of coumarin-6-loaded PCL NPs and TPGS- b -PCL NPs after incubated with HeLa cells. Notes: ( A ) Uptake efficiency of coumarin-6-loaded PCL NPs and TPGS- b -PCL NPs by HeLa cells (* P
    Figure Legend Snippet: Cellular uptake of coumarin-6-loaded PCL NPs and TPGS- b -PCL NPs after incubated with HeLa cells. Notes: ( A ) Uptake efficiency of coumarin-6-loaded PCL NPs and TPGS- b -PCL NPs by HeLa cells (* P

    Techniques Used: Incubation

    Characterization of TPGS-b-PCL copolymer. Notes: ( A ) Typical gel permeation chromatograms of TPGS and TPGS- b -PCL diblock copolymer; ( B ) thermogravimetric profiles of TPGS and TPGS- b -PCL copolymer. Abbreviations: min, minutes; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.
    Figure Legend Snippet: Characterization of TPGS-b-PCL copolymer. Notes: ( A ) Typical gel permeation chromatograms of TPGS and TPGS- b -PCL diblock copolymer; ( B ) thermogravimetric profiles of TPGS and TPGS- b -PCL copolymer. Abbreviations: min, minutes; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.

    Techniques Used:

    In vitro release profiles of genistein-loaded PCL NPs and TPGS- b -PCL NPs. Abbreviations: NPs, nanoparticles; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.
    Figure Legend Snippet: In vitro release profiles of genistein-loaded PCL NPs and TPGS- b -PCL NPs. Abbreviations: NPs, nanoparticles; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.

    Techniques Used: In Vitro

    Typical 1 H-NMR spectra of TPGS- b -PCL copolymer ( A ) and TPGS ( B ). Abbreviations: 1 H-NMR, proton nuclear magnetic resonance; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.
    Figure Legend Snippet: Typical 1 H-NMR spectra of TPGS- b -PCL copolymer ( A ) and TPGS ( B ). Abbreviations: 1 H-NMR, proton nuclear magnetic resonance; PCL, poly(ε-caprolactone); TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.

    Techniques Used: Nuclear Magnetic Resonance

    FESEM image ( A ), TEM image ( B ), and DLS spectra ( C ) of genistein-loaded TPGS- b -PCL NPs. Abbreviations: DLS, dynamic light scattering; FESEM, field emission scanning electron microscopic; NPs, nanoparticles; PCL, poly(ε-caprolactone); TEM, transmission electron microscopic; TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.
    Figure Legend Snippet: FESEM image ( A ), TEM image ( B ), and DLS spectra ( C ) of genistein-loaded TPGS- b -PCL NPs. Abbreviations: DLS, dynamic light scattering; FESEM, field emission scanning electron microscopic; NPs, nanoparticles; PCL, poly(ε-caprolactone); TEM, transmission electron microscopic; TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.

    Techniques Used: Transmission Electron Microscopy, Transmission Assay

    Antitumor effect of genistein formulated in TPGS- b -PCL in comparison with pristine genistein and genistein-loaded PCL NPs (n=5). Notes: ( A ) Tumor growth curve of the BALB/c nude mice bearing HeLa cells xenograft after administration (* P
    Figure Legend Snippet: Antitumor effect of genistein formulated in TPGS- b -PCL in comparison with pristine genistein and genistein-loaded PCL NPs (n=5). Notes: ( A ) Tumor growth curve of the BALB/c nude mice bearing HeLa cells xenograft after administration (* P

    Techniques Used: Mouse Assay

    28) Product Images from "An Artifact in LC-MS/MS Measurement of Glutamine and Glutamic Acid: In-Source Cyclization to Pyroglutamic Acid"

    Article Title: An Artifact in LC-MS/MS Measurement of Glutamine and Glutamic Acid: In-Source Cyclization to Pyroglutamic Acid

    Journal: Analytical Chemistry

    doi: 10.1021/ac501451v

    (A) Mechanism of Gln and Glu cyclization to pGlu after ammonia and water loss, respectively. (B) MRMs of Glu, Gln, and pGlu from an equimolar mix of Glu, Gln, and pGlu standards (1 mM) showing the “in-source” formation of pGlu from Gln and Glu. The MS spectra were acquired using the optimal conditions for each analyte as described in the Experimental Section .
    Figure Legend Snippet: (A) Mechanism of Gln and Glu cyclization to pGlu after ammonia and water loss, respectively. (B) MRMs of Glu, Gln, and pGlu from an equimolar mix of Glu, Gln, and pGlu standards (1 mM) showing the “in-source” formation of pGlu from Gln and Glu. The MS spectra were acquired using the optimal conditions for each analyte as described in the Experimental Section .

    Techniques Used: Mass Spectrometry

    (A) Effect of fragmentor voltage (FV) on peak ratio (signal intensity of pGlu from Gln /signal intensity of Gln) at different concentrations of Gln (0.39–200 μM). Peak ratio increased on increasing FV above 100 V. Gln signal was not quantifiable above FV 120 V. (B) MRM traces acquired using 200 μM Gln at FV 76 V (optimal for Gln), FV 120 V, and FV 200 V, illustrating the loss of Gln signal as FV is increased above 100 V.
    Figure Legend Snippet: (A) Effect of fragmentor voltage (FV) on peak ratio (signal intensity of pGlu from Gln /signal intensity of Gln) at different concentrations of Gln (0.39–200 μM). Peak ratio increased on increasing FV above 100 V. Gln signal was not quantifiable above FV 120 V. (B) MRM traces acquired using 200 μM Gln at FV 76 V (optimal for Gln), FV 120 V, and FV 200 V, illustrating the loss of Gln signal as FV is increased above 100 V.

    Techniques Used:

    29) Product Images from "Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice"

    Article Title: Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.012237

    Differentiation and behavioural assays of non-db- and db-derived BM macrophages. (A) Representative bright-field images of nondb- and db-derived BM macrophages after 7 days culture in M-CSF (scale bar: 25 μm). (B) Representative flow cytometry plots of non-dband db-derived BM macrophages after 7 days culture in M-CSF, showing expression of macrophage markers CD11b and F4.80. (C) qRTPCR analysis of myeloid cell markers Emr1 (F4.80, left panel), Ly6g (Gr-1, centre panel) and Itgam (CD11b, right panel) at day 1 and day 7 of culture in M-CSF in non-db (grey bars) and db (black bars) BM macrophages from three independent RNA isolations for each condition. (D) qRT-PCR analysis of CCR2 expression in nondb (grey bar) and db (black bar) BM macrophages after 7 days of culture in M-CSF from three independent RNA isolations for each condition. (E) Chemotactic response to MCP-1 in a transwell assay showing mean number of cells per field of view (FoV) of non-db and db BM macrophages from three independent experiments. (F) Scratch wound migration assay of non-db (grey diamonds) and db (black squares) BM macrophages showing mean number of cells migrated into scratch wound at 4-hour intervals over 16 hours from three independent experiments. (G) Adhesion assay of non-db (grey bar) and db (black bar) BM macrophages showing mean number of cells per field of view adhered onto activated endothelial cells after 4 hours from three independent experiments. (H) Adhesion assays comparing classically activated (CA) and alternatively activated (AA) BM macrophages with wound-derived macrophages at days 3 and 7 following wounding ( n =3 for each condition).
    Figure Legend Snippet: Differentiation and behavioural assays of non-db- and db-derived BM macrophages. (A) Representative bright-field images of nondb- and db-derived BM macrophages after 7 days culture in M-CSF (scale bar: 25 μm). (B) Representative flow cytometry plots of non-dband db-derived BM macrophages after 7 days culture in M-CSF, showing expression of macrophage markers CD11b and F4.80. (C) qRTPCR analysis of myeloid cell markers Emr1 (F4.80, left panel), Ly6g (Gr-1, centre panel) and Itgam (CD11b, right panel) at day 1 and day 7 of culture in M-CSF in non-db (grey bars) and db (black bars) BM macrophages from three independent RNA isolations for each condition. (D) qRT-PCR analysis of CCR2 expression in nondb (grey bar) and db (black bar) BM macrophages after 7 days of culture in M-CSF from three independent RNA isolations for each condition. (E) Chemotactic response to MCP-1 in a transwell assay showing mean number of cells per field of view (FoV) of non-db and db BM macrophages from three independent experiments. (F) Scratch wound migration assay of non-db (grey diamonds) and db (black squares) BM macrophages showing mean number of cells migrated into scratch wound at 4-hour intervals over 16 hours from three independent experiments. (G) Adhesion assay of non-db (grey bar) and db (black bar) BM macrophages showing mean number of cells per field of view adhered onto activated endothelial cells after 4 hours from three independent experiments. (H) Adhesion assays comparing classically activated (CA) and alternatively activated (AA) BM macrophages with wound-derived macrophages at days 3 and 7 following wounding ( n =3 for each condition).

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Transwell Assay, Migration, Cell Adhesion Assay

    30) Product Images from "Modulation of RNase E Activity by Alternative RNA Binding Sites"

    Article Title: Modulation of RNase E Activity by Alternative RNA Binding Sites

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090610

    Identification of a hypoactive N-Rne mutant. (A) Location of the isolated single amino-acid substitutions in the crystal structure of the N-terminal region of RNase E. Two tryptic peptides that were UV-crosslinked to p-BR13, 24 LYDLDIESPGHEQK 37 and 65 HGFLPLK 71 , are colored in blue and green, respectively. p-BR13 is colored in yellow. The diagram was generated using PyMOL software. (B) Tandem mass spectrum assigned to the predicted b - and y -ions generated by collision-induced fragmentation of the peptide, 24 L Y C DLDIESPGHEQK 37 , with the Y25 residue bound to cytosine ( m/z = 629.63, z = +3, mass error = −1.20 ppm). (C) Tandem mass spectrum assigned to the predicted b - and y -ions generated from collision-induced fragmentation of the peptide, 24 LYDLDIESPGHE Q A K 37 , with the Q36 residue bound to adenine ( m/z = 637.64, z = +3, mass error = 8.62 ppm). (D) Growth characteristics of cells expressing wild-type N-Rne or the Q36R or Y25A mutant proteins. Growth of KSL2000 cells harboring pNRNE4, pNRNE4-Q36R, or pNRNE4-Y25A was measured individually on LB-agar plates containing 1.0 to 1000 µM IPTG. KSL2000 harboring pACYC177 grew only when full-length RNase E was expressed from pBAD-RNE in the presence of 0.2% arabinose. Numbers on the top indicate the number of bacterial cells in each spot.
    Figure Legend Snippet: Identification of a hypoactive N-Rne mutant. (A) Location of the isolated single amino-acid substitutions in the crystal structure of the N-terminal region of RNase E. Two tryptic peptides that were UV-crosslinked to p-BR13, 24 LYDLDIESPGHEQK 37 and 65 HGFLPLK 71 , are colored in blue and green, respectively. p-BR13 is colored in yellow. The diagram was generated using PyMOL software. (B) Tandem mass spectrum assigned to the predicted b - and y -ions generated by collision-induced fragmentation of the peptide, 24 L Y C DLDIESPGHEQK 37 , with the Y25 residue bound to cytosine ( m/z = 629.63, z = +3, mass error = −1.20 ppm). (C) Tandem mass spectrum assigned to the predicted b - and y -ions generated from collision-induced fragmentation of the peptide, 24 LYDLDIESPGHE Q A K 37 , with the Q36 residue bound to adenine ( m/z = 637.64, z = +3, mass error = 8.62 ppm). (D) Growth characteristics of cells expressing wild-type N-Rne or the Q36R or Y25A mutant proteins. Growth of KSL2000 cells harboring pNRNE4, pNRNE4-Q36R, or pNRNE4-Y25A was measured individually on LB-agar plates containing 1.0 to 1000 µM IPTG. KSL2000 harboring pACYC177 grew only when full-length RNase E was expressed from pBAD-RNE in the presence of 0.2% arabinose. Numbers on the top indicate the number of bacterial cells in each spot.

    Techniques Used: Mutagenesis, Isolation, Generated, Software, Expressing

    Effects of Y25A and Q36R on the catalytic activity of RNase E in vivo and in vitro . (A) Plasmid copy number of pNRNE4, pNRNE4-Q36R and pNRNE4-Y25A in KSL2000. Plasmids were purified from KSL2000 cells harboring pNRNE4, pNRNE4-Q36R or pNRNE4-Y25A and were digested with Hin dIII, which has a unique cleavage site in all of the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pBAD-RNE), which replicates independently of Rne, by measuring the molar ratio of the ColE1-type plasmid to the pBAD-RNE plasmid. (B) Growth characteristics of KSL2003 cells expressing wild-type N-Rne or the Q36R or Y25A mutant proteins. Growth of KSL2003 cells harboring pLAC-RNE2, pLAC-RNE2-Q36R, or pLAC-RNE2-Y25A was measured individually on LB-agar plates containing 1.0 to 1000 µM IPTG. Numbers on the top indicate the number of bacterial cells in each spot. (C) Plasmid copy number of pET28a in KSL2003. Plasmids were purified from KSL2003, KSL2003-Q36R or KSL2003-Y25A cells harboring pET28a and digested with Hin dIII, which has a unique cleavage site in all the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pLAC-RNE2, pLAC-RNE2-Q36R or pLAC-RNE2-Y25A) by measuring the molar ratio of the ColE1-type plasmid to the pSC101-derived plasmid. (D) Expression profiles of Rne and mutant proteins in KSL2003. The membrane probed with an anti-Rne polyclonal antibody was stripped and reprobed with an anti-S1 polyclonal antibody to provide an internal standard. The relative abundance of protein was quantified by setting the amount of wild-type Rne to 1. KSL2003 cells were grown in LB medium containing 10 µM IPTG. (E) In vitro cleavage of p-BR13 by wild-type N-Rne, Q36R and Y25A mutant proteins. Two pmol of 5′ end-labeled p-BR13 was incubated with 1 pmol of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of cleavage buffer at 37°C. Samples were removed at each indicated time point and mixed with an equal volume of loading buffer. Samples were denatured at 65°C for 5 min and loaded onto 15% polyacrylamide gel containing 8 M urea. The radioactivity in each band was quantified using a phosphorimager and OptiQuant software.
    Figure Legend Snippet: Effects of Y25A and Q36R on the catalytic activity of RNase E in vivo and in vitro . (A) Plasmid copy number of pNRNE4, pNRNE4-Q36R and pNRNE4-Y25A in KSL2000. Plasmids were purified from KSL2000 cells harboring pNRNE4, pNRNE4-Q36R or pNRNE4-Y25A and were digested with Hin dIII, which has a unique cleavage site in all of the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pBAD-RNE), which replicates independently of Rne, by measuring the molar ratio of the ColE1-type plasmid to the pBAD-RNE plasmid. (B) Growth characteristics of KSL2003 cells expressing wild-type N-Rne or the Q36R or Y25A mutant proteins. Growth of KSL2003 cells harboring pLAC-RNE2, pLAC-RNE2-Q36R, or pLAC-RNE2-Y25A was measured individually on LB-agar plates containing 1.0 to 1000 µM IPTG. Numbers on the top indicate the number of bacterial cells in each spot. (C) Plasmid copy number of pET28a in KSL2003. Plasmids were purified from KSL2003, KSL2003-Q36R or KSL2003-Y25A cells harboring pET28a and digested with Hin dIII, which has a unique cleavage site in all the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pLAC-RNE2, pLAC-RNE2-Q36R or pLAC-RNE2-Y25A) by measuring the molar ratio of the ColE1-type plasmid to the pSC101-derived plasmid. (D) Expression profiles of Rne and mutant proteins in KSL2003. The membrane probed with an anti-Rne polyclonal antibody was stripped and reprobed with an anti-S1 polyclonal antibody to provide an internal standard. The relative abundance of protein was quantified by setting the amount of wild-type Rne to 1. KSL2003 cells were grown in LB medium containing 10 µM IPTG. (E) In vitro cleavage of p-BR13 by wild-type N-Rne, Q36R and Y25A mutant proteins. Two pmol of 5′ end-labeled p-BR13 was incubated with 1 pmol of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of cleavage buffer at 37°C. Samples were removed at each indicated time point and mixed with an equal volume of loading buffer. Samples were denatured at 65°C for 5 min and loaded onto 15% polyacrylamide gel containing 8 M urea. The radioactivity in each band was quantified using a phosphorimager and OptiQuant software.

    Techniques Used: Activity Assay, In Vivo, In Vitro, Plasmid Preparation, Purification, Expressing, Mutagenesis, Derivative Assay, Labeling, Incubation, Radioactivity, Software

    Effects of mutant proteins on RNA binding activity. (A, B) Electrophoretic mobility shift assay. The 5′ end labeled p-BR13 (0.5 pmol) was incubated with increasing concentrations of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of EMSA buffer, incubated on ice (A) or at room temperature (B) for 10 min, and analyzed by 12% nondenaturing PAGE. Binding constants were calculated based on slopes calculated from the graph. To avoid induction of RNA cleavage, Mg 2+ was omitted from the EMSA reactions. (C) UV crosslinking of N-Rne-wt, N-Rne-Q36R and N-Rne-Y25A to p-BR13. Two pmol of p-BR13 was incubated with 100 pmol of N-wild-type Rne, Q36R or Y25A mutant protein in 20 µl of crosslinking buffer and exposed to UV light for 30 min. Samples were loaded onto 10% polyacrylamide gels (lanes 2, 5, 8) and samples in the absence of p-BR13 (lane 1, 4, 7) or UV irradiation (lanes 3, 6, 9) were also loaded as controls. The gel was stained with Coomassie brilliant blue and dried. The radioactivity in each band was detected using a phosphorimager and OptiQuant software. The number of crosslinked p-BR13 per pmol of protein was calculated.
    Figure Legend Snippet: Effects of mutant proteins on RNA binding activity. (A, B) Electrophoretic mobility shift assay. The 5′ end labeled p-BR13 (0.5 pmol) was incubated with increasing concentrations of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of EMSA buffer, incubated on ice (A) or at room temperature (B) for 10 min, and analyzed by 12% nondenaturing PAGE. Binding constants were calculated based on slopes calculated from the graph. To avoid induction of RNA cleavage, Mg 2+ was omitted from the EMSA reactions. (C) UV crosslinking of N-Rne-wt, N-Rne-Q36R and N-Rne-Y25A to p-BR13. Two pmol of p-BR13 was incubated with 100 pmol of N-wild-type Rne, Q36R or Y25A mutant protein in 20 µl of crosslinking buffer and exposed to UV light for 30 min. Samples were loaded onto 10% polyacrylamide gels (lanes 2, 5, 8) and samples in the absence of p-BR13 (lane 1, 4, 7) or UV irradiation (lanes 3, 6, 9) were also loaded as controls. The gel was stained with Coomassie brilliant blue and dried. The radioactivity in each band was detected using a phosphorimager and OptiQuant software. The number of crosslinked p-BR13 per pmol of protein was calculated.

    Techniques Used: Mutagenesis, RNA Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Purification, Polyacrylamide Gel Electrophoresis, Binding Assay, Irradiation, Staining, Radioactivity, Software

    31) Product Images from "Laser Capture Microdissection of Pancreatic Acinar Cells to Identify Proteomic Alterations in a Murine Model of Caerulein-Induced Pancreatitis"

    Article Title: Laser Capture Microdissection of Pancreatic Acinar Cells to Identify Proteomic Alterations in a Murine Model of Caerulein-Induced Pancreatitis

    Journal: Clinical and Translational Gastroenterology

    doi: 10.1038/ctg.2017.15

    Comparison of 6-week caerulein mouse proteomic data vs. human proteomic data that characterized chronic pancreatitis using liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis of proteins retrieved from formalin-fixed paraffin-embedded (FFPE) biopsies. The correlation between human and mouse protein expression was assessed by generation of Pearson's correlation coefficient using a comparison (of the log 2) of ratios of pancreatitis vs. normal expression of all common proteins in the two data sets. A simple linear regression line was fitted to the data points.
    Figure Legend Snippet: Comparison of 6-week caerulein mouse proteomic data vs. human proteomic data that characterized chronic pancreatitis using liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis of proteins retrieved from formalin-fixed paraffin-embedded (FFPE) biopsies. The correlation between human and mouse protein expression was assessed by generation of Pearson's correlation coefficient using a comparison (of the log 2) of ratios of pancreatitis vs. normal expression of all common proteins in the two data sets. A simple linear regression line was fitted to the data points.

    Techniques Used: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Formalin-fixed Paraffin-Embedded, Expressing

    32) Product Images from "The action of β-hydroxybutyrate on the growth, metabolism and global histone H3 acetylation of spontaneous mouse mammary tumours: evidence of a β-hydroxybutyrate paradox"

    Article Title: The action of β-hydroxybutyrate on the growth, metabolism and global histone H3 acetylation of spontaneous mouse mammary tumours: evidence of a β-hydroxybutyrate paradox

    Journal: Cancer & Metabolism

    doi: 10.1186/s40170-017-0166-z

    Ki-67 (proliferation marker, panels a and b ), CD31 (measure of vascularity, panels c and d ) and cleaved caspase 3 (apoptosis marker, panels e and f ) immunohistochemical staining of tumour sections at day 21 from control mice ( left ) and mice treated with β-OHB (500 mg/kg), injected ip daily for 3 weeks ( right ) ( Bars show 100 μm). See Table 1 for quantitative analysis
    Figure Legend Snippet: Ki-67 (proliferation marker, panels a and b ), CD31 (measure of vascularity, panels c and d ) and cleaved caspase 3 (apoptosis marker, panels e and f ) immunohistochemical staining of tumour sections at day 21 from control mice ( left ) and mice treated with β-OHB (500 mg/kg), injected ip daily for 3 weeks ( right ) ( Bars show 100 μm). See Table 1 for quantitative analysis

    Techniques Used: Marker, Immunohistochemistry, Staining, Mouse Assay, Injection

    Effect of β-OHB (0, 0.5, 5, 10 mM) on glucose consumption and lactate production (μmol/10 6 cells/24 h) at 24 h when MMTV-NEU-NT cells were incubated in low-glucose (1 g/L) DMEM. N = 3, ** p
    Figure Legend Snippet: Effect of β-OHB (0, 0.5, 5, 10 mM) on glucose consumption and lactate production (μmol/10 6 cells/24 h) at 24 h when MMTV-NEU-NT cells were incubated in low-glucose (1 g/L) DMEM. N = 3, ** p

    Techniques Used: Incubation

    a Tumour volume in MMTV-NEU-NT tumour-bearing control mice and mice treated with β-OHB (500 mg/kg), injected ip daily for 3 weeks. Control: N = 25 tumours (18 mice), Treated: N = 23 tumours (19 mice); * p
    Figure Legend Snippet: a Tumour volume in MMTV-NEU-NT tumour-bearing control mice and mice treated with β-OHB (500 mg/kg), injected ip daily for 3 weeks. Control: N = 25 tumours (18 mice), Treated: N = 23 tumours (19 mice); * p

    Techniques Used: Mouse Assay, Injection

    Metabolites (μmol/g) measured by 1 H MRS in tumour extracts at day 21 from MMTV-NEU-NT tumour-bearing control mice and mice treated with β-OHB (500 mg/kg), injected ip daily for 3 weeks. a Branched chain amino acids, glutamine and glycine,** p
    Figure Legend Snippet: Metabolites (μmol/g) measured by 1 H MRS in tumour extracts at day 21 from MMTV-NEU-NT tumour-bearing control mice and mice treated with β-OHB (500 mg/kg), injected ip daily for 3 weeks. a Branched chain amino acids, glutamine and glycine,** p

    Techniques Used: Mouse Assay, Injection

    Phosphorus containing metabolites (μmol/g), measured by 31 P MRS, in tumour extracts at day 21 from MMTV-NEU-NT tumour-bearing control mice and mice treated with β-OHB (500 mg/kg), injected ip daily for 3 weeks. Phosphomonoesters (PME = phosphocholine (PC) + phosphoethanolamine (PE)), phosphodiesters (PDE = glycerophosphocholine + glycerophosphoethanolamine), inorganic phosphate (Pi), and β-ATP/Pi. Control: N = 8 to 13 (6 mice); Treated: N = 13–18 (12 mice), * p ≤ 0.05
    Figure Legend Snippet: Phosphorus containing metabolites (μmol/g), measured by 31 P MRS, in tumour extracts at day 21 from MMTV-NEU-NT tumour-bearing control mice and mice treated with β-OHB (500 mg/kg), injected ip daily for 3 weeks. Phosphomonoesters (PME = phosphocholine (PC) + phosphoethanolamine (PE)), phosphodiesters (PDE = glycerophosphocholine + glycerophosphoethanolamine), inorganic phosphate (Pi), and β-ATP/Pi. Control: N = 8 to 13 (6 mice); Treated: N = 13–18 (12 mice), * p ≤ 0.05

    Techniques Used: Mouse Assay, Injection

    Metabolites (μmol/g) measured by 1 H MRS in liver extracts at day 21 from MMTV-NEU-NT tumour-bearing control mice and mice treated with β-OHB (500 mg/kg) injected ip daily for 3 weeks. Isoleucine, valine, glutamine, glycine, lactate, glucose, ATP + ADP, choline, phosphocholine (PC), glycerophosphocholine (GPC), creatine (Cr), phosphocreatine (PCr), myo-inositol. Control: N = 12; Treated: N = 8; * p
    Figure Legend Snippet: Metabolites (μmol/g) measured by 1 H MRS in liver extracts at day 21 from MMTV-NEU-NT tumour-bearing control mice and mice treated with β-OHB (500 mg/kg) injected ip daily for 3 weeks. Isoleucine, valine, glutamine, glycine, lactate, glucose, ATP + ADP, choline, phosphocholine (PC), glycerophosphocholine (GPC), creatine (Cr), phosphocreatine (PCr), myo-inositol. Control: N = 12; Treated: N = 8; * p

    Techniques Used: Mouse Assay, Injection, Gel Permeation Chromatography, Polymerase Chain Reaction

    33) Product Images from "Novel and shared neoantigen derived from histone 3 variant H3.3K27M mutation for glioma T cell therapy"

    Article Title: Novel and shared neoantigen derived from histone 3 variant H3.3K27M mutation for glioma T cell therapy

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20171046

    The H3.3K27M peptide is detectable by LC-MS/MS in the HLA class I immunopeptidome of glioma cells bearing the H3.3K27M mutation . HLA class I peptides were biochemically purified from U87H3.3K27M glioma cells and analyzed by LC-MS/MS with a synthetic heavy version of the H3.3K27M peptide as the reference. (A) U87H3.3K27M HLA class I immunopeptidome shows two coeluting isotope patterns corresponding to the target m / z and mass difference of the oxidized forms of the heavy and the endogenous H3.3K27M peptides. (B) Fragmentation spectrum of the heavy peak, showing identification of the oxidized heavy H3.3K27M peptide. (C) Zoom-in of the light isotope pattern shows m / z values and distances between peaks as expected from the endogenous H3.3K27M peptide. This experiment was conducted once.
    Figure Legend Snippet: The H3.3K27M peptide is detectable by LC-MS/MS in the HLA class I immunopeptidome of glioma cells bearing the H3.3K27M mutation . HLA class I peptides were biochemically purified from U87H3.3K27M glioma cells and analyzed by LC-MS/MS with a synthetic heavy version of the H3.3K27M peptide as the reference. (A) U87H3.3K27M HLA class I immunopeptidome shows two coeluting isotope patterns corresponding to the target m / z and mass difference of the oxidized forms of the heavy and the endogenous H3.3K27M peptides. (B) Fragmentation spectrum of the heavy peak, showing identification of the oxidized heavy H3.3K27M peptide. (C) Zoom-in of the light isotope pattern shows m / z values and distances between peaks as expected from the endogenous H3.3K27M peptide. This experiment was conducted once.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Mutagenesis, Purification

    34) Product Images from "The epigenetic reader SntB regulates secondary metabolism, development and global histone modifications in Aspergillus flavus"

    Article Title: The epigenetic reader SntB regulates secondary metabolism, development and global histone modifications in Aspergillus flavus

    Journal: Fungal genetics and biology : FG & B

    doi: 10.1016/j.fgb.2018.08.004

    Deletion of sntB leads to a greater change in secondary metabolite profile than overexpression. Metabolites were extracted from twelve-day-old cultures on PDA, run on a UHPLC-HRMS, and analyzed via XCMS. Experiment was completed in triplicate. A) Comparison of metabolite extracts from sntB deletion and wild type. Each dot represents a peak called by XCMS. The (–)log10 of the p value is plotted on the y-axis, with a gray dashed line indicating where the p value is equal to 0.05, values higher on the y-axis indicating higher statistical significance. Log2 of the fold change is on the x-axis, with values in the right half more abundant in the deletion strain, and values on the left half more abundant in the wild type. Red dots indicate known final products that were detected by the program including aflavarin, aflatoxin, asparasone A, ditryptophenaline, and leporin B. B) Same analysis comparing the overexpression of sntB to wild type.
    Figure Legend Snippet: Deletion of sntB leads to a greater change in secondary metabolite profile than overexpression. Metabolites were extracted from twelve-day-old cultures on PDA, run on a UHPLC-HRMS, and analyzed via XCMS. Experiment was completed in triplicate. A) Comparison of metabolite extracts from sntB deletion and wild type. Each dot represents a peak called by XCMS. The (–)log10 of the p value is plotted on the y-axis, with a gray dashed line indicating where the p value is equal to 0.05, values higher on the y-axis indicating higher statistical significance. Log2 of the fold change is on the x-axis, with values in the right half more abundant in the deletion strain, and values on the left half more abundant in the wild type. Red dots indicate known final products that were detected by the program including aflavarin, aflatoxin, asparasone A, ditryptophenaline, and leporin B. B) Same analysis comparing the overexpression of sntB to wild type.

    Techniques Used: Over Expression

    35) Product Images from "Quantum Dot-Based Molecular Beacon to Monitor Intracellular MicroRNAs"

    Article Title: Quantum Dot-Based Molecular Beacon to Monitor Intracellular MicroRNAs

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s150612872

    ( A ) Quenching efficiency of the R9-QD-miR124a beacons in the tube. A fixed concentration of the QDs (10 pM) were conjugated with various concentrations (0–300 pM) of the miR124a-targeting oligomer to determine the optimal concentration of miR124a needed to achieve the best quenching effect. The fluorescence activity of the R9-QD-miR124a beacons gradually decreased as the concentration of miR124a-targeting oligomer increased. ROI analysis from the fluorescence tube image showed that the fluorescence signal decreased in a dose-dependent manner ( * p
    Figure Legend Snippet: ( A ) Quenching efficiency of the R9-QD-miR124a beacons in the tube. A fixed concentration of the QDs (10 pM) were conjugated with various concentrations (0–300 pM) of the miR124a-targeting oligomer to determine the optimal concentration of miR124a needed to achieve the best quenching effect. The fluorescence activity of the R9-QD-miR124a beacons gradually decreased as the concentration of miR124a-targeting oligomer increased. ROI analysis from the fluorescence tube image showed that the fluorescence signal decreased in a dose-dependent manner ( * p

    Techniques Used: Concentration Assay, Fluorescence, Activity Assay

    The activation of fluorescence intensity of the R9-QD-miR124a beacon in C6 cells. ( A ) In vitro cytotoxicity of QDs and the R9-QD-miR124a beacons in cells; ( B ) R9 peptide conjugation improved internalization efficiency and miR124a specificity of the R9-QD-miR124a beacons in C6 cells. The fluorescence signal from the quenched R9-QD-miR124a beacons was activated in the exogenous miR124a-treated group (300 pM), unlike in the mutant-treated group (300 pM); ( C ) The visualization of fluorescence recovery of the R9-QD-miR124a beacons in C6 cells. The R9-QD-miR124a beacons were incubated with C6 cells for 1 h 30 min, and exogenous miR124a or the miR124a mutant was added to the pre-treated C6 cells. Confocal microscopy imaging showed that the fluorescence signal of the R9-QD-miR124a beacons was significantly activated in C6 cells after treatment with exogenous miR124a. In contrast, induction of the miR124a mutant showed a weak fluorescence signal of the R9-QD-miR124a beacon in C6 cells, suggesting maintenance of the quenched fluorescence signal.
    Figure Legend Snippet: The activation of fluorescence intensity of the R9-QD-miR124a beacon in C6 cells. ( A ) In vitro cytotoxicity of QDs and the R9-QD-miR124a beacons in cells; ( B ) R9 peptide conjugation improved internalization efficiency and miR124a specificity of the R9-QD-miR124a beacons in C6 cells. The fluorescence signal from the quenched R9-QD-miR124a beacons was activated in the exogenous miR124a-treated group (300 pM), unlike in the mutant-treated group (300 pM); ( C ) The visualization of fluorescence recovery of the R9-QD-miR124a beacons in C6 cells. The R9-QD-miR124a beacons were incubated with C6 cells for 1 h 30 min, and exogenous miR124a or the miR124a mutant was added to the pre-treated C6 cells. Confocal microscopy imaging showed that the fluorescence signal of the R9-QD-miR124a beacons was significantly activated in C6 cells after treatment with exogenous miR124a. In contrast, induction of the miR124a mutant showed a weak fluorescence signal of the R9-QD-miR124a beacon in C6 cells, suggesting maintenance of the quenched fluorescence signal.

    Techniques Used: Activation Assay, Fluorescence, In Vitro, Conjugation Assay, Mutagenesis, Incubation, Confocal Microscopy, Imaging

    Characterization of the R9-QD-miR124a beacons. ( A ) Transmission electron microscopy (TEM) images; ( B ) electrophoretic shift assay; and ( C ) dynamic light scattering (DLS) analysis of the unconjugated QDs and the R9-QD-miR124a beacons; ( D ) Coupling efficiencies of the miR124a-targeting oligomer and the R9 peptide to QDs.
    Figure Legend Snippet: Characterization of the R9-QD-miR124a beacons. ( A ) Transmission electron microscopy (TEM) images; ( B ) electrophoretic shift assay; and ( C ) dynamic light scattering (DLS) analysis of the unconjugated QDs and the R9-QD-miR124a beacons; ( D ) Coupling efficiencies of the miR124a-targeting oligomer and the R9 peptide to QDs.

    Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Shift Assay

    36) Product Images from "Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity *"

    Article Title: Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M115.055780

    HLA-DR ligandome analysis and characteristics. A , Workflow for the analysis of the HLA-DR-presented ligandome and proteome of MUTZ-3 DC. For the ligandome, following affinity purification of HLA-DR-peptide complexes, peptides were released by acid elution, fractionated by strong cation exchange (SCX) and analyzed by LC-MS/MS using ETD, EThCD, and HCD fragmentation. Global proteome analysis was performed following trypsin digestion of lysed MUTZ-3 cells, using SCX fractionation prior to LC-MS/MS analysis. B , Venn diagram displaying the overlap and unique contribution to the identified HLA-DR ligandome by EThcD, ETD and HCD. C , Peptide length distribution in the combined HLA-DR ligandome ( n = 13,918). D , Illustrative nested set of HLA-DR-associated peptides derived from Elongation factor 1-α 1, consisting of 10 peptide length variants with a consensus core binding motif but varying extended N- and C termini.
    Figure Legend Snippet: HLA-DR ligandome analysis and characteristics. A , Workflow for the analysis of the HLA-DR-presented ligandome and proteome of MUTZ-3 DC. For the ligandome, following affinity purification of HLA-DR-peptide complexes, peptides were released by acid elution, fractionated by strong cation exchange (SCX) and analyzed by LC-MS/MS using ETD, EThCD, and HCD fragmentation. Global proteome analysis was performed following trypsin digestion of lysed MUTZ-3 cells, using SCX fractionation prior to LC-MS/MS analysis. B , Venn diagram displaying the overlap and unique contribution to the identified HLA-DR ligandome by EThcD, ETD and HCD. C , Peptide length distribution in the combined HLA-DR ligandome ( n = 13,918). D , Illustrative nested set of HLA-DR-associated peptides derived from Elongation factor 1-α 1, consisting of 10 peptide length variants with a consensus core binding motif but varying extended N- and C termini.

    Techniques Used: Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Fractionation, Derivative Assay, Binding Assay

    37) Product Images from "Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors"

    Article Title: Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors

    Journal: Theranostics

    doi: 10.7150/thno.13069

    TEM images and schematic representation of MnMEIO NPs conjugated with (right) or without (left) mPEG, CyTE777 and the bispecific antibody. The core size of MnMEIO NPs was 9.1 ± 0.3 nm. Then, the organic and aqueous dispersions of nanoparticles were also shown.
    Figure Legend Snippet: TEM images and schematic representation of MnMEIO NPs conjugated with (right) or without (left) mPEG, CyTE777 and the bispecific antibody. The core size of MnMEIO NPs was 9.1 ± 0.3 nm. Then, the organic and aqueous dispersions of nanoparticles were also shown.

    Techniques Used: Transmission Electron Microscopy

    38) Product Images from "Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia"

    Article Title: Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia

    Journal: Scientific Reports

    doi: 10.1038/srep06048

    Sequence abnormalities in B117H Ara-C resistant cells verified by Sanger sequencing. (a) RNA-seq reads were mapped to the NCBI reference genome. Visualization of Dck expression using IGV. (b) Transcripts were assembled independently of a reference transcriptome using Cufflinks, then mapped to the mm9 mouse genome using Cuffcompare, and the resulting gtf file was visualized by IGV. (c) Sanger sequencing of DNA in B117H cells identified an 878 nt deletion spanning the splice acceptor of intron 6 and the translated portion of exon 7. Sanger sequencing of RNA verified a transcript matching the configuration identified by TopHat and IGV.
    Figure Legend Snippet: Sequence abnormalities in B117H Ara-C resistant cells verified by Sanger sequencing. (a) RNA-seq reads were mapped to the NCBI reference genome. Visualization of Dck expression using IGV. (b) Transcripts were assembled independently of a reference transcriptome using Cufflinks, then mapped to the mm9 mouse genome using Cuffcompare, and the resulting gtf file was visualized by IGV. (c) Sanger sequencing of DNA in B117H cells identified an 878 nt deletion spanning the splice acceptor of intron 6 and the translated portion of exon 7. Sanger sequencing of RNA verified a transcript matching the configuration identified by TopHat and IGV.

    Techniques Used: Sequencing, Acetylene Reduction Assay, RNA Sequencing Assay, Expressing

    Reducing Dck expression in the B117P parental cell line results in increases of the IC 50 value for Ara-C. (a) Schematic of the CRISPR-Cas9 cloning vector. (b) MTS-tetrazolium assays confirmed a shift in response to Ara-C in cells transfected with 2 different gRNAs ( Dck CKO-1 and Dck CKO-2) using the CRISPR system. (c) Reduction of Dck expression in B117P cells was accomplished marginally by RNA interference (KD1 and KD2) or completely using TALENs (KO). Error bars depict range. (d) Structure of the TALENs used to knockout Dck in the B117P cell line. (e) DNA modifications to Dck in the TALEN based KO cell lines confirmed a 1 nt deletion in the T2A cells, a 32 nt deletion in the T6B cells, and a 2 nt deletion in the T11A cells, and in each case the deletion occurred just a little over 50 nt from the translation start site. (f) RT-PCR of cDNA copy of RNA of a 595 base sequence straddling the translational start site. Primers are described in Supplementary Table S11 . (g) Western blot verifying the absence of Dck proteins in the Dck KO clones. Cropped images presented in figure. Full-length blots can be found in Supplementary Figure S6b .
    Figure Legend Snippet: Reducing Dck expression in the B117P parental cell line results in increases of the IC 50 value for Ara-C. (a) Schematic of the CRISPR-Cas9 cloning vector. (b) MTS-tetrazolium assays confirmed a shift in response to Ara-C in cells transfected with 2 different gRNAs ( Dck CKO-1 and Dck CKO-2) using the CRISPR system. (c) Reduction of Dck expression in B117P cells was accomplished marginally by RNA interference (KD1 and KD2) or completely using TALENs (KO). Error bars depict range. (d) Structure of the TALENs used to knockout Dck in the B117P cell line. (e) DNA modifications to Dck in the TALEN based KO cell lines confirmed a 1 nt deletion in the T2A cells, a 32 nt deletion in the T6B cells, and a 2 nt deletion in the T11A cells, and in each case the deletion occurred just a little over 50 nt from the translation start site. (f) RT-PCR of cDNA copy of RNA of a 595 base sequence straddling the translational start site. Primers are described in Supplementary Table S11 . (g) Western blot verifying the absence of Dck proteins in the Dck KO clones. Cropped images presented in figure. Full-length blots can be found in Supplementary Figure S6b .

    Techniques Used: Expressing, Acetylene Reduction Assay, CRISPR, Clone Assay, Plasmid Preparation, Transfection, TALENs, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot

    39) Product Images from "Antitumor effect of antitissue factor antibody‐MMAE conjugate in human pancreatic tumor xenografts"

    Article Title: Antitumor effect of antitissue factor antibody‐MMAE conjugate in human pancreatic tumor xenografts

    Journal: International Journal of Cancer

    doi: 10.1002/ijc.29492

    Preparation and characterization of ADCs. ( a ) Structure of ADC. Approximately three molecules of MMAE were conjugated to one antibody by a valine‐citrulline dipeptide linker. ( b ) Particle size of mAbs and ADCs. The mean particle sizes of mAbs were 9.5 nm (anti‐human mAb) and 7.6 nm (anti‐mouse mAb) and the mean particle sizes of ADCs were 12.0 nm (anti‐human ADC) and 12.6 nm (anti‐mouse ADC). ( c ) SDS‐PAGE of mAbs and ADCs. M: molecular markers. D) Releasing of MMAE from ADCs. The rates of free MMAE released from the anti‐human and ‐mouse ADCs in an acidic buffer with cathepsin B at 37 °C for 24 hr was 72.2% and 70.8%, respectively. None of the free MMAE was released from both the anti‐human and ‐mouse ADCs in a neutral buffer without cathepsin B at 37 °C for up to 48 hr.
    Figure Legend Snippet: Preparation and characterization of ADCs. ( a ) Structure of ADC. Approximately three molecules of MMAE were conjugated to one antibody by a valine‐citrulline dipeptide linker. ( b ) Particle size of mAbs and ADCs. The mean particle sizes of mAbs were 9.5 nm (anti‐human mAb) and 7.6 nm (anti‐mouse mAb) and the mean particle sizes of ADCs were 12.0 nm (anti‐human ADC) and 12.6 nm (anti‐mouse ADC). ( c ) SDS‐PAGE of mAbs and ADCs. M: molecular markers. D) Releasing of MMAE from ADCs. The rates of free MMAE released from the anti‐human and ‐mouse ADCs in an acidic buffer with cathepsin B at 37 °C for 24 hr was 72.2% and 70.8%, respectively. None of the free MMAE was released from both the anti‐human and ‐mouse ADCs in a neutral buffer without cathepsin B at 37 °C for up to 48 hr.

    Techniques Used: SDS Page

    Affinity and Internalization of ADCs. ( a ) TF expression in the four human pancreatic cancer cell lines. Relative TF expression in BxPC‐3, PSN‐1, Capan‐1 and Panc‐1, normalized by a negative control, were 244.2, 45.2, 7.8 and 3.7, respectively, thus showing that BxPC‐3 cells had a high TF expression, PSN‐1 had a moderate TF expression and Capan‐1 and Panc‐1 had low TF expression. ( b ) Affinity of mAbs and ADCs to pancreatic cancer cells. Anti‐human mAb and anti‐human ADC were reacted to BxPC‐3 cells. Meanwhile anti‐mouse mAb, anti‐mouse ADC, control mAb and control ADC did not recognize human cells. ( c ) Internalization of ADC. The anti‐human ADC was sufficiently internalized into the cytoplasm after a 3‐hr incubation at 37°C and localized in a lysosome. Anti‐human ADC and lysosomes were indicated red and green, respectively. Colocalization of anti‐human ADC and lysosomes was merged as yellow. Scale bar represents 10 μm.
    Figure Legend Snippet: Affinity and Internalization of ADCs. ( a ) TF expression in the four human pancreatic cancer cell lines. Relative TF expression in BxPC‐3, PSN‐1, Capan‐1 and Panc‐1, normalized by a negative control, were 244.2, 45.2, 7.8 and 3.7, respectively, thus showing that BxPC‐3 cells had a high TF expression, PSN‐1 had a moderate TF expression and Capan‐1 and Panc‐1 had low TF expression. ( b ) Affinity of mAbs and ADCs to pancreatic cancer cells. Anti‐human mAb and anti‐human ADC were reacted to BxPC‐3 cells. Meanwhile anti‐mouse mAb, anti‐mouse ADC, control mAb and control ADC did not recognize human cells. ( c ) Internalization of ADC. The anti‐human ADC was sufficiently internalized into the cytoplasm after a 3‐hr incubation at 37°C and localized in a lysosome. Anti‐human ADC and lysosomes were indicated red and green, respectively. Colocalization of anti‐human ADC and lysosomes was merged as yellow. Scale bar represents 10 μm.

    Techniques Used: Expressing, Negative Control, Incubation

    40) Product Images from "Module-detection approaches for the integration of multilevel omics data highlight the comprehensive response of Aspergillus fumigatus to caspofungin"

    Article Title: Module-detection approaches for the integration of multilevel omics data highlight the comprehensive response of Aspergillus fumigatus to caspofungin

    Journal: BMC Systems Biology

    doi: 10.1186/s12918-018-0620-8

    Caspofungin-induced increased production of the secondary metabolite fumagillin. LC-ESI-ITMS extracted ion chromatograms (EIC) at m/z 459.0–459.4 amu (left), HPLC-UV/PDA chromatograms (center) and UV/PDA spectra at RT = 13.67 min (right) of 250 μg/ml fumagillin reference standard (top) and crude extract of A. fumigatus without (center) and with caspofungin treatment (bottom)
    Figure Legend Snippet: Caspofungin-induced increased production of the secondary metabolite fumagillin. LC-ESI-ITMS extracted ion chromatograms (EIC) at m/z 459.0–459.4 amu (left), HPLC-UV/PDA chromatograms (center) and UV/PDA spectra at RT = 13.67 min (right) of 250 μg/ml fumagillin reference standard (top) and crude extract of A. fumigatus without (center) and with caspofungin treatment (bottom)

    Techniques Used: High Performance Liquid Chromatography

    41) Product Images from "Racemization at the Asp 58 residue in αA‐crystallin from the lens of high myopic cataract patients"

    Article Title: Racemization at the Asp 58 residue in αA‐crystallin from the lens of high myopic cataract patients

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13363

    Protein purification and identification of αA‐crystallin. ( A ) Elution profiles of water‐soluble factions of age‐related cataract ( ARC ) lens proteins using gel filtration chromatography. ( B ) Elution profiles of water‐soluble factions of high myopic cataract ( HMC ) lens proteins using gel filtration chromatography. All of the lens protein fraction samples of Peak 1, 2 and 3 (arrows) in Fig. 1 A and B were collected for further analysis to identify αA‐crystallin. ( C ) Lens protein fraction analysis by Western blotting. The existence of αA‐crystallin was confirmed mainly in Peak 1.
    Figure Legend Snippet: Protein purification and identification of αA‐crystallin. ( A ) Elution profiles of water‐soluble factions of age‐related cataract ( ARC ) lens proteins using gel filtration chromatography. ( B ) Elution profiles of water‐soluble factions of high myopic cataract ( HMC ) lens proteins using gel filtration chromatography. All of the lens protein fraction samples of Peak 1, 2 and 3 (arrows) in Fig. 1 A and B were collected for further analysis to identify αA‐crystallin. ( C ) Lens protein fraction analysis by Western blotting. The existence of αA‐crystallin was confirmed mainly in Peak 1.

    Techniques Used: Protein Purification, Filtration, Chromatography, Western Blot

    ( A ) Bar diagram showing the amount of Asp 58 racemization in αA‐crystallin of age‐related cataract ( ARC ) and high myopic cataract ( HMC ) lenses. Compared with ARC lenses, HMC lenses showed a significant decrease in the amount of l ‐isoAsp 58 ( P
    Figure Legend Snippet: ( A ) Bar diagram showing the amount of Asp 58 racemization in αA‐crystallin of age‐related cataract ( ARC ) and high myopic cataract ( HMC ) lenses. Compared with ARC lenses, HMC lenses showed a significant decrease in the amount of l ‐isoAsp 58 ( P

    Techniques Used:

    Scatter plot showing the amount of Asp58 racemization in αA‐crystallin for each sample from the age‐related cataract ( ARC ) and high myopic cataract ( HMC ) groups. ( A ) Statistical analysis showed a significantly negative correlation between axial lengths and the racemization level of l ‐isoAsp 58 (Spearman ρ = −0.699, P = 0.011). ( B ) There was a significantly positive correlation between axial lengths and the racemization level of d ‐Asp 58 (Spearman ρ = 0.839, P = 0.001). ( C ) No significant correlation was detected between axial lengths and the amount of l ‐Asp 58in ARC and HMC lenses ( P > 0.05). ( D ) No significant correlation was detected between axial lengths and the amount of d ‐isoAsp 58 in ARC and HMC lenses ( P > 0.05).
    Figure Legend Snippet: Scatter plot showing the amount of Asp58 racemization in αA‐crystallin for each sample from the age‐related cataract ( ARC ) and high myopic cataract ( HMC ) groups. ( A ) Statistical analysis showed a significantly negative correlation between axial lengths and the racemization level of l ‐isoAsp 58 (Spearman ρ = −0.699, P = 0.011). ( B ) There was a significantly positive correlation between axial lengths and the racemization level of d ‐Asp 58 (Spearman ρ = 0.839, P = 0.001). ( C ) No significant correlation was detected between axial lengths and the amount of l ‐Asp 58in ARC and HMC lenses ( P > 0.05). ( D ) No significant correlation was detected between axial lengths and the amount of d ‐isoAsp 58 in ARC and HMC lenses ( P > 0.05).

    Techniques Used:

    Identification of Asp isomers in αA‐crystallin. ( A ) Representative LC ‐ MS / MS trace showing the separation of the four Asp isoforms of the αA‐crystallin tryptic peptide (55–65) TVLDSGISEVR . Peptides containing d ‐Asp, d ‐isoAsp, l ‐Asp or l ‐isoAsp at position 58 were synthesized. To measure racemization in αA‐crystallin, all forms of the peptide were summed and modifications for each were expressed as a % of the total peak area. ( B ) Representative graphs showing the separation of the four Asp 58 isoforms in αA‐crystallin extracted from age‐related cataract ( ARC ) lenses. In this ARC case, the ratio of four distinct isomeric forms was l ‐isoAsp21.79%, d ‐Asp 29.22%, l ‐Asp 16.58% and d ‐isoAsp 32.40%.( C ) Representative graphs showing the separation of the four Asp 58 isoforms in αA‐crystallin extracted from high myopic cataract ( HMC ) lenses. In this HMC case, the ratio of four distinct isomeric forms was l ‐isoAsp17.74%, d ‐Asp 41.68%, l ‐Asp 14.04% and d ‐isoAsp 26.54%.
    Figure Legend Snippet: Identification of Asp isomers in αA‐crystallin. ( A ) Representative LC ‐ MS / MS trace showing the separation of the four Asp isoforms of the αA‐crystallin tryptic peptide (55–65) TVLDSGISEVR . Peptides containing d ‐Asp, d ‐isoAsp, l ‐Asp or l ‐isoAsp at position 58 were synthesized. To measure racemization in αA‐crystallin, all forms of the peptide were summed and modifications for each were expressed as a % of the total peak area. ( B ) Representative graphs showing the separation of the four Asp 58 isoforms in αA‐crystallin extracted from age‐related cataract ( ARC ) lenses. In this ARC case, the ratio of four distinct isomeric forms was l ‐isoAsp21.79%, d ‐Asp 29.22%, l ‐Asp 16.58% and d ‐isoAsp 32.40%.( C ) Representative graphs showing the separation of the four Asp 58 isoforms in αA‐crystallin extracted from high myopic cataract ( HMC ) lenses. In this HMC case, the ratio of four distinct isomeric forms was l ‐isoAsp17.74%, d ‐Asp 41.68%, l ‐Asp 14.04% and d ‐isoAsp 26.54%.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Synthesized

    42) Product Images from "P-cadherin signals through the laminin receptor ?6?4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells"

    Article Title: P-cadherin signals through the laminin receptor ?6?4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells

    Journal: Oncotarget

    doi:

    P-cadherin controls the expression of α6β4 integrin heterodimer in basal-like breast cancer cell lines Transient inhibition of the P-cadherin/CDH3 gene in MDA-MB-468 and BT-20 cells leads to a decrease in the expression of α6 and β4 integrin subunits, measured by western blot (A); the mRNA level of α6 integrin/ITGA6 is decreased upon P-cadherin silencing, whereas β4 integrin/ITGB4 mRNA level is unaffected (B); Co-immunoprecipitation experiments show that P-cadherin directly interacts with the β4 integrin subunit, but not with the α6 integrin subunit in these cells (the BT-20 cell line is represented).
    Figure Legend Snippet: P-cadherin controls the expression of α6β4 integrin heterodimer in basal-like breast cancer cell lines Transient inhibition of the P-cadherin/CDH3 gene in MDA-MB-468 and BT-20 cells leads to a decrease in the expression of α6 and β4 integrin subunits, measured by western blot (A); the mRNA level of α6 integrin/ITGA6 is decreased upon P-cadherin silencing, whereas β4 integrin/ITGB4 mRNA level is unaffected (B); Co-immunoprecipitation experiments show that P-cadherin directly interacts with the β4 integrin subunit, but not with the α6 integrin subunit in these cells (the BT-20 cell line is represented).

    Techniques Used: Expressing, Inhibition, Multiple Displacement Amplification, Western Blot, Immunoprecipitation

    43) Product Images from "Antitumor effect of antitissue factor antibody‐MMAE conjugate in human pancreatic tumor xenografts"

    Article Title: Antitumor effect of antitissue factor antibody‐MMAE conjugate in human pancreatic tumor xenografts

    Journal: International Journal of Cancer

    doi: 10.1002/ijc.29492

    Preparation and characterization of ADCs. ( a ) Structure of ADC. Approximately three molecules of MMAE were conjugated to one antibody by a valine‐citrulline dipeptide linker. ( b ) Particle size of mAbs and ADCs. The mean particle sizes of mAbs were 9.5 nm (anti‐human mAb) and 7.6 nm (anti‐mouse mAb) and the mean particle sizes of ADCs were 12.0 nm (anti‐human ADC) and 12.6 nm (anti‐mouse ADC). ( c ) SDS‐PAGE of mAbs and ADCs. M: molecular markers. D) Releasing of MMAE from ADCs. The rates of free MMAE released from the anti‐human and ‐mouse ADCs in an acidic buffer with cathepsin B at 37 °C for 24 hr was 72.2% and 70.8%, respectively. None of the free MMAE was released from both the anti‐human and ‐mouse ADCs in a neutral buffer without cathepsin B at 37 °C for up to 48 hr.
    Figure Legend Snippet: Preparation and characterization of ADCs. ( a ) Structure of ADC. Approximately three molecules of MMAE were conjugated to one antibody by a valine‐citrulline dipeptide linker. ( b ) Particle size of mAbs and ADCs. The mean particle sizes of mAbs were 9.5 nm (anti‐human mAb) and 7.6 nm (anti‐mouse mAb) and the mean particle sizes of ADCs were 12.0 nm (anti‐human ADC) and 12.6 nm (anti‐mouse ADC). ( c ) SDS‐PAGE of mAbs and ADCs. M: molecular markers. D) Releasing of MMAE from ADCs. The rates of free MMAE released from the anti‐human and ‐mouse ADCs in an acidic buffer with cathepsin B at 37 °C for 24 hr was 72.2% and 70.8%, respectively. None of the free MMAE was released from both the anti‐human and ‐mouse ADCs in a neutral buffer without cathepsin B at 37 °C for up to 48 hr.

    Techniques Used: SDS Page

    44) Product Images from "Interactome analyses revealed that the U1 snRNP machinery overlaps extensively with the RNAP II machinery and contains multiple ALS/SMA-causative proteins"

    Article Title: Interactome analyses revealed that the U1 snRNP machinery overlaps extensively with the RNAP II machinery and contains multiple ALS/SMA-causative proteins

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27136-3

    Top hit interactors in the FET proteins and MATR3 interactomes. The top ranked (by total peptide number) proteins in each interactome are shown. The rank, HGNC official symbol, calculated molecular weight, best-known function, total and unique peptide counts are shown. Functions in splicing (pink), transcription (txn, orange), DNA damage response (green), neuronal (blue) and other (black) are indicated. The symbols of ALS-causative proteins are in red. The stars indicate U1 snRNP components.
    Figure Legend Snippet: Top hit interactors in the FET proteins and MATR3 interactomes. The top ranked (by total peptide number) proteins in each interactome are shown. The rank, HGNC official symbol, calculated molecular weight, best-known function, total and unique peptide counts are shown. Functions in splicing (pink), transcription (txn, orange), DNA damage response (green), neuronal (blue) and other (black) are indicated. The symbols of ALS-causative proteins are in red. The stars indicate U1 snRNP components.

    Techniques Used: Molecular Weight

    Protein-protein interaction network of the U1 snRNP machinery. Same as Fig. 3 , except that the network of the U1 snRNP interactome is shown.
    Figure Legend Snippet: Protein-protein interaction network of the U1 snRNP machinery. Same as Fig. 3 , except that the network of the U1 snRNP interactome is shown.

    Techniques Used:

    The U1 snRNP machinery overlaps with the RNAP II machinery. ( a ) Venn diagram showing overlap of the U1 snRNP and RNAP II machineries. ( b ) HeLa cell nuclear extract was separated on a Sephacryl-S500 column. The indicated fractions were used for Western analyses with antibodies against RNAP II and U1 snRNP components (SNRPA and SNRPC). Fraction 25 is the void volume and 69 is the included volume. ( c ) IPs were carried out from nuclear extract using an antibody to the POLR2A subunit of the RNAP II (left panel) or an antibody to the SNRPC subunit of the U1 snRNP (right panel) as well as a negative control antibody (EIF4A3) followed by Westerns with antibodies to the DEAD box helicases (DHX9, DDX5 and DDX17). ( d ) IPs were carried out with the indicated antibodies from nuclear extract treated with a U1 snRNA AMO or a negative control AMO followed by Western using the SNRPC antibody.
    Figure Legend Snippet: The U1 snRNP machinery overlaps with the RNAP II machinery. ( a ) Venn diagram showing overlap of the U1 snRNP and RNAP II machineries. ( b ) HeLa cell nuclear extract was separated on a Sephacryl-S500 column. The indicated fractions were used for Western analyses with antibodies against RNAP II and U1 snRNP components (SNRPA and SNRPC). Fraction 25 is the void volume and 69 is the included volume. ( c ) IPs were carried out from nuclear extract using an antibody to the POLR2A subunit of the RNAP II (left panel) or an antibody to the SNRPC subunit of the U1 snRNP (right panel) as well as a negative control antibody (EIF4A3) followed by Westerns with antibodies to the DEAD box helicases (DHX9, DDX5 and DDX17). ( d ) IPs were carried out with the indicated antibodies from nuclear extract treated with a U1 snRNA AMO or a negative control AMO followed by Western using the SNRPC antibody.

    Techniques Used: Western Blot, Negative Control

    FET proteins and MATR3 associate with U1 snRNP. ( a ) Immunoprecipitations (IPs) were carried out with antibodies to FET proteins or MATR3 followed by analysis on a Coomassie-stained gel. Molecular weight markers and protein identified by mass spectrometry are indicated. ( b ) IPs were carried out from nuclear extract using a negative control antibody (EIF4A3) or an antibody to the SNRPC subunit of the U1 snRNP followed by Westerns with the indicated antibodies. ( c ) IPs were carried out with the indicated antibodies from nuclear extract treated with a U1 snRNA AMO or a negative control AMO followed by Western using the SNRPC antibody. ( d ) Same as ( c ) except that total RNAs from the IPs were examined on a denaturing gel stained with ethidium bromide.
    Figure Legend Snippet: FET proteins and MATR3 associate with U1 snRNP. ( a ) Immunoprecipitations (IPs) were carried out with antibodies to FET proteins or MATR3 followed by analysis on a Coomassie-stained gel. Molecular weight markers and protein identified by mass spectrometry are indicated. ( b ) IPs were carried out from nuclear extract using a negative control antibody (EIF4A3) or an antibody to the SNRPC subunit of the U1 snRNP followed by Westerns with the indicated antibodies. ( c ) IPs were carried out with the indicated antibodies from nuclear extract treated with a U1 snRNA AMO or a negative control AMO followed by Western using the SNRPC antibody. ( d ) Same as ( c ) except that total RNAs from the IPs were examined on a denaturing gel stained with ethidium bromide.

    Techniques Used: Staining, Molecular Weight, Mass Spectrometry, Negative Control, Western Blot

    45) Product Images from "P-cadherin signals through the laminin receptor ?6?4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells"

    Article Title: P-cadherin signals through the laminin receptor ?6?4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells

    Journal: Oncotarget

    doi:

    P-cadherin controls the expression of α6β4 integrin heterodimer in basal-like breast cancer cell lines Transient inhibition of the P-cadherin/CDH3 gene in MDA-MB-468 and BT-20 cells leads to a decrease in the expression of α6 and β4 integrin subunits, measured by western blot (A); the mRNA level of α6 integrin/ITGA6 is decreased upon P-cadherin silencing, whereas β4 integrin/ITGB4 mRNA level is unaffected (B); Co-immunoprecipitation experiments show that P-cadherin directly interacts with the β4 integrin subunit, but not with the α6 integrin subunit in these cells (the BT-20 cell line is represented).
    Figure Legend Snippet: P-cadherin controls the expression of α6β4 integrin heterodimer in basal-like breast cancer cell lines Transient inhibition of the P-cadherin/CDH3 gene in MDA-MB-468 and BT-20 cells leads to a decrease in the expression of α6 and β4 integrin subunits, measured by western blot (A); the mRNA level of α6 integrin/ITGA6 is decreased upon P-cadherin silencing, whereas β4 integrin/ITGB4 mRNA level is unaffected (B); Co-immunoprecipitation experiments show that P-cadherin directly interacts with the β4 integrin subunit, but not with the α6 integrin subunit in these cells (the BT-20 cell line is represented).

    Techniques Used: Expressing, Inhibition, Multiple Displacement Amplification, Western Blot, Immunoprecipitation

    46) Product Images from "Disinfectant Performance of a Chlorine Regenerable Antibacterial Microfiber Fabric as a Reusable Wiper"

    Article Title: Disinfectant Performance of a Chlorine Regenerable Antibacterial Microfiber Fabric as a Reusable Wiper

    Journal: Materials

    doi: 10.3390/ma12010127

    ( A ) FTIR-ATR spectra of the fabric after 5 recharging cycles: ( a ) pristine fabric, ( b ) after cleaning 150 tables, ( c ) difference spectrum of subtracting (a) from (b), ( B ) SEM image of the fabric after 5 recharging cycles.
    Figure Legend Snippet: ( A ) FTIR-ATR spectra of the fabric after 5 recharging cycles: ( a ) pristine fabric, ( b ) after cleaning 150 tables, ( c ) difference spectrum of subtracting (a) from (b), ( B ) SEM image of the fabric after 5 recharging cycles.

    Techniques Used:

    Stability analysis: ( A ) FTIR-ATR spectra of ( a ) pristine fabric, ( b ) grafted fabric, ( c ) ten times washed grafted fabric, and ( d ) difference spectrum, subtracting (a) from (c); ( B ) Scanning electron microscope (SEM) micrographs of ( a ) pristine fabric, ( b ) grafted fabric, ( c ) ten times washed grafted fabric; ( C ) Washing stability of grafted fabrics. Symbol: Mean ± SD.
    Figure Legend Snippet: Stability analysis: ( A ) FTIR-ATR spectra of ( a ) pristine fabric, ( b ) grafted fabric, ( c ) ten times washed grafted fabric, and ( d ) difference spectrum, subtracting (a) from (c); ( B ) Scanning electron microscope (SEM) micrographs of ( a ) pristine fabric, ( b ) grafted fabric, ( c ) ten times washed grafted fabric; ( C ) Washing stability of grafted fabrics. Symbol: Mean ± SD.

    Techniques Used: Microscopy

    47) Product Images from "Hypobaric hypoxia induced renal damage is mediated by altering redox pathway"

    Article Title: Hypobaric hypoxia induced renal damage is mediated by altering redox pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0195701

    Network analysis and gene ontology annotations of the proteins identified by MALDI-TOF MS. A) Network analysis of identified proteins with their predicted interactions using STRING db 10.0 software. B) Gene ontology classifications (Pie chart analysis) of proteins were obtained by using PANTHER 10.0 software and proteins were distributed according to their Biological Process, Molecular Function, Cellular Component and Pathway Involved.
    Figure Legend Snippet: Network analysis and gene ontology annotations of the proteins identified by MALDI-TOF MS. A) Network analysis of identified proteins with their predicted interactions using STRING db 10.0 software. B) Gene ontology classifications (Pie chart analysis) of proteins were obtained by using PANTHER 10.0 software and proteins were distributed according to their Biological Process, Molecular Function, Cellular Component and Pathway Involved.

    Techniques Used: Mass Spectrometry, Software

    48) Product Images from "The epigenetic reader SntB regulates secondary metabolism, development and global histone modifications in Aspergillus flavus"

    Article Title: The epigenetic reader SntB regulates secondary metabolism, development and global histone modifications in Aspergillus flavus

    Journal: Fungal genetics and biology : FG & B

    doi: 10.1016/j.fgb.2018.08.004

    Deletion of sntB leads to a greater change in secondary metabolite profile than overexpression. Metabolites were extracted from twelve-day-old cultures on PDA, run on a UHPLC-HRMS, and analyzed via XCMS. Experiment was completed in triplicate. A) Comparison of metabolite extracts from sntB deletion and wild type. Each dot represents a peak called by XCMS. The (–)log10 of the p value is plotted on the y-axis, with a gray dashed line indicating where the p value is equal to 0.05, values higher on the y-axis indicating higher statistical significance. Log2 of the fold change is on the x-axis, with values in the right half more abundant in the deletion strain, and values on the left half more abundant in the wild type. Red dots indicate known final products that were detected by the program including aflavarin, aflatoxin, asparasone A, ditryptophenaline, and leporin B. B) Same analysis comparing the overexpression of sntB to wild type.
    Figure Legend Snippet: Deletion of sntB leads to a greater change in secondary metabolite profile than overexpression. Metabolites were extracted from twelve-day-old cultures on PDA, run on a UHPLC-HRMS, and analyzed via XCMS. Experiment was completed in triplicate. A) Comparison of metabolite extracts from sntB deletion and wild type. Each dot represents a peak called by XCMS. The (–)log10 of the p value is plotted on the y-axis, with a gray dashed line indicating where the p value is equal to 0.05, values higher on the y-axis indicating higher statistical significance. Log2 of the fold change is on the x-axis, with values in the right half more abundant in the deletion strain, and values on the left half more abundant in the wild type. Red dots indicate known final products that were detected by the program including aflavarin, aflatoxin, asparasone A, ditryptophenaline, and leporin B. B) Same analysis comparing the overexpression of sntB to wild type.

    Techniques Used: Over Expression

    49) Product Images from "Cholesterol biosynthesis pathway as a novel mechanism of resistance to estrogen deprivation in estrogen receptor-positive breast cancer"

    Article Title: Cholesterol biosynthesis pathway as a novel mechanism of resistance to estrogen deprivation in estrogen receptor-positive breast cancer

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-016-0713-5

    25-HC and 27-HC enhance recruitment of ER to endogenous E-regulated genes in ER+ LTED cells. wt-MCF7 and MCF7 LTED cells were treated with E2, 25-HC and 27-HC alone or in combination with ICI182780 ( ICI ) for 24 hours. mRNA was extracted and quantitative RT-PCR used to measure expression of TFF1 . a Data shown are representative of three independent biological experiments. Bars : ± SEM. b MCF7 LTED cells were synchronized using α-amanitin and treated with 25-HC for 45 minutes [ 26 ]. ChIP was carried out to assess the recruitment of ER to the GREB1 and TFF1 promoters, respectively. To provide evidence for an activated complex, histone deacetylase CBP recruitment was also assessed. c Effect of 27-HC on ER and CBP recruitment to the TFF1 and GREB1 promoters. Data shown are representative of three technical replicates. Bars : ± SEM. DCC Dextran-coated charcoal, E2 estradiol, ER estrogen receptor alpha, ERE estrogen response elements, HC hydroxycholesterol, LTED , long-term estrogen deprivation, wt wild type
    Figure Legend Snippet: 25-HC and 27-HC enhance recruitment of ER to endogenous E-regulated genes in ER+ LTED cells. wt-MCF7 and MCF7 LTED cells were treated with E2, 25-HC and 27-HC alone or in combination with ICI182780 ( ICI ) for 24 hours. mRNA was extracted and quantitative RT-PCR used to measure expression of TFF1 . a Data shown are representative of three independent biological experiments. Bars : ± SEM. b MCF7 LTED cells were synchronized using α-amanitin and treated with 25-HC for 45 minutes [ 26 ]. ChIP was carried out to assess the recruitment of ER to the GREB1 and TFF1 promoters, respectively. To provide evidence for an activated complex, histone deacetylase CBP recruitment was also assessed. c Effect of 27-HC on ER and CBP recruitment to the TFF1 and GREB1 promoters. Data shown are representative of three technical replicates. Bars : ± SEM. DCC Dextran-coated charcoal, E2 estradiol, ER estrogen receptor alpha, ERE estrogen response elements, HC hydroxycholesterol, LTED , long-term estrogen deprivation, wt wild type

    Techniques Used: Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, Histone Deacetylase Assay, Droplet Countercurrent Chromatography

    50) Product Images from "C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae"

    Article Title: C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007341

    C-terminal modification of VesB. VesB was overexpressed and purified from supernatants of LB-grown  V .  cholerae  culture in the presence of protease inhibitors at 4° C. The purified material was subjected to intact mass analysis (A) and SDS-PAGE, in-gel trypsin digestion and LC-MS/MS analysis (B-D).  A. Left panel:  Deconvoluted ESI mass spectrum indicates four molecular masses of VesB. The masses highlighted with asterisks correspond to theoretical VesB masses generated through cleavage at either Ser 378  ( * ) or Ser 380  ( ** ) (see inset with C-terminal sequence of VesB). The relative amount of the four VesB species varied between three separate purifications. Accuracy of the instrument: 0.01% of molecular mass.  Right panel:  The deconvoluted mass spectrum of protein eluting at a higher acetonitrile concentration.  B.  C-terminal peptides of VesB generated by trypsin and identified by LC-MS/MS analysis. The asterisks correspond to cleavage at Ser 378  ( * ) and Ser 380  ( ** ).  C.  Representative MS/MS spectrum for the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions.  D.  MS/MS spectrum for the VesB peptide IQLDTSPFAPSASSGG with a C-terminal 197 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da).  E.  VesB was extracted from WT  V .  cholerae  with Triton X-100, purified on benzamidine sepharose and subjected to SDS-PAGE, in-gel trypsin digestion and LC -MS/MS analysis. Representative MS/MS spectrum of the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions.  F.  MS/MS spectrum of the 197-Da modified peptide IQLDTSPFAPSASSGG as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da).  G.  VesB is attached to a glycerophosphoethanolamine containing moiety, possibly phosphatidylethanolamine, via its C-terminal glycine. The red and blue arrows indicate possible sites of hydrolysis or fragmentation, respectively, resulting in VesB species with either ethanolamine or glycerophosphoethanolamine.
    Figure Legend Snippet: C-terminal modification of VesB. VesB was overexpressed and purified from supernatants of LB-grown V . cholerae culture in the presence of protease inhibitors at 4° C. The purified material was subjected to intact mass analysis (A) and SDS-PAGE, in-gel trypsin digestion and LC-MS/MS analysis (B-D). A. Left panel: Deconvoluted ESI mass spectrum indicates four molecular masses of VesB. The masses highlighted with asterisks correspond to theoretical VesB masses generated through cleavage at either Ser 378 ( * ) or Ser 380 ( ** ) (see inset with C-terminal sequence of VesB). The relative amount of the four VesB species varied between three separate purifications. Accuracy of the instrument: 0.01% of molecular mass. Right panel: The deconvoluted mass spectrum of protein eluting at a higher acetonitrile concentration. B. C-terminal peptides of VesB generated by trypsin and identified by LC-MS/MS analysis. The asterisks correspond to cleavage at Ser 378 ( * ) and Ser 380 ( ** ). C. Representative MS/MS spectrum for the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. D. MS/MS spectrum for the VesB peptide IQLDTSPFAPSASSGG with a C-terminal 197 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da). E. VesB was extracted from WT V . cholerae with Triton X-100, purified on benzamidine sepharose and subjected to SDS-PAGE, in-gel trypsin digestion and LC -MS/MS analysis. Representative MS/MS spectrum of the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. F. MS/MS spectrum of the 197-Da modified peptide IQLDTSPFAPSASSGG as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da). G. VesB is attached to a glycerophosphoethanolamine containing moiety, possibly phosphatidylethanolamine, via its C-terminal glycine. The red and blue arrows indicate possible sites of hydrolysis or fragmentation, respectively, resulting in VesB species with either ethanolamine or glycerophosphoethanolamine.

    Techniques Used: Modification, Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Generated, Sequencing, Concentration Assay

    51) Product Images from "C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae"

    Article Title: C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007341

    C-terminal modification of VesB. VesB was overexpressed and purified from supernatants of LB-grown  V .  cholerae  culture in the presence of protease inhibitors at 4° C. The purified material was subjected to intact mass analysis (A) and SDS-PAGE, in-gel trypsin digestion and LC-MS/MS analysis (B-D).  A. Left panel:  Deconvoluted ESI mass spectrum indicates four molecular masses of VesB. The masses highlighted with asterisks correspond to theoretical VesB masses generated through cleavage at either Ser 378  ( * ) or Ser 380  ( ** ) (see inset with C-terminal sequence of VesB). The relative amount of the four VesB species varied between three separate purifications. Accuracy of the instrument: 0.01% of molecular mass.  Right panel:  The deconvoluted mass spectrum of protein eluting at a higher acetonitrile concentration.  B.  C-terminal peptides of VesB generated by trypsin and identified by LC-MS/MS analysis. The asterisks correspond to cleavage at Ser 378  ( * ) and Ser 380  ( ** ).  C.  Representative MS/MS spectrum for the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions.  D.  MS/MS spectrum for the VesB peptide IQLDTSPFAPSASSGG with a C-terminal 197 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da).  E.  VesB was extracted from WT  V .  cholerae  with Triton X-100, purified on benzamidine sepharose and subjected to SDS-PAGE, in-gel trypsin digestion and LC -MS/MS analysis. Representative MS/MS spectrum of the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions.  F.  MS/MS spectrum of the 197-Da modified peptide IQLDTSPFAPSASSGG as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da).  G.  VesB is attached to a glycerophosphoethanolamine containing moiety, possibly phosphatidylethanolamine, via its C-terminal glycine. The red and blue arrows indicate possible sites of hydrolysis or fragmentation, respectively, resulting in VesB species with either ethanolamine or glycerophosphoethanolamine.
    Figure Legend Snippet: C-terminal modification of VesB. VesB was overexpressed and purified from supernatants of LB-grown V . cholerae culture in the presence of protease inhibitors at 4° C. The purified material was subjected to intact mass analysis (A) and SDS-PAGE, in-gel trypsin digestion and LC-MS/MS analysis (B-D). A. Left panel: Deconvoluted ESI mass spectrum indicates four molecular masses of VesB. The masses highlighted with asterisks correspond to theoretical VesB masses generated through cleavage at either Ser 378 ( * ) or Ser 380 ( ** ) (see inset with C-terminal sequence of VesB). The relative amount of the four VesB species varied between three separate purifications. Accuracy of the instrument: 0.01% of molecular mass. Right panel: The deconvoluted mass spectrum of protein eluting at a higher acetonitrile concentration. B. C-terminal peptides of VesB generated by trypsin and identified by LC-MS/MS analysis. The asterisks correspond to cleavage at Ser 378 ( * ) and Ser 380 ( ** ). C. Representative MS/MS spectrum for the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. D. MS/MS spectrum for the VesB peptide IQLDTSPFAPSASSGG with a C-terminal 197 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da). E. VesB was extracted from WT V . cholerae with Triton X-100, purified on benzamidine sepharose and subjected to SDS-PAGE, in-gel trypsin digestion and LC -MS/MS analysis. Representative MS/MS spectrum of the C-terminal peptide IQLDTSPFAPSASSGG of VesB shows modification of the C-terminal glycine with a 43 Da moiety as evidenced by the presence of the y1–ion and indicated subsequent y-ions. F. MS/MS spectrum of the 197-Da modified peptide IQLDTSPFAPSASSGG as evidenced by the presence of the y1–ion and indicated subsequent y-ions as well as the two fragment-ions y 7 -172 at m/z 587.3 and y 10 -172 at m/z 904.2 generated by neutral loss of the phosphoglyceryl moiety (C 3 H 9 O 6 P, 172.013 Da). G. VesB is attached to a glycerophosphoethanolamine containing moiety, possibly phosphatidylethanolamine, via its C-terminal glycine. The red and blue arrows indicate possible sites of hydrolysis or fragmentation, respectively, resulting in VesB species with either ethanolamine or glycerophosphoethanolamine.

    Techniques Used: Modification, Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Generated, Sequencing, Concentration Assay

    52) Product Images from "Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice"

    Article Title: Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.012237

    Differentiation and behavioural assays of non-db- and db-derived BM macrophages. (A) Representative bright-field images of nondb- and db-derived BM macrophages after 7 days culture in M-CSF (scale bar: 25 μm). (B) Representative flow cytometry plots of non-dband db-derived BM macrophages after 7 days culture in M-CSF, showing expression of macrophage markers CD11b and F4.80. (C) qRTPCR analysis of myeloid cell markers Emr1 (F4.80, left panel), Ly6g (Gr-1, centre panel) and Itgam (CD11b, right panel) at day 1 and day 7 of culture in M-CSF in non-db (grey bars) and db (black bars) BM macrophages from three independent RNA isolations for each condition. (D) qRT-PCR analysis of CCR2 expression in nondb (grey bar) and db (black bar) BM macrophages after 7 days of culture in M-CSF from three independent RNA isolations for each condition. (E) Chemotactic response to MCP-1 in a transwell assay showing mean number of cells per field of view (FoV) of non-db and db BM macrophages from three independent experiments. (F) Scratch wound migration assay of non-db (grey diamonds) and db (black squares) BM macrophages showing mean number of cells migrated into scratch wound at 4-hour intervals over 16 hours from three independent experiments. (G) Adhesion assay of non-db (grey bar) and db (black bar) BM macrophages showing mean number of cells per field of view adhered onto activated endothelial cells after 4 hours from three independent experiments. (H) Adhesion assays comparing classically activated (CA) and alternatively activated (AA) BM macrophages with wound-derived macrophages at days 3 and 7 following wounding ( n =3 for each condition).
    Figure Legend Snippet: Differentiation and behavioural assays of non-db- and db-derived BM macrophages. (A) Representative bright-field images of nondb- and db-derived BM macrophages after 7 days culture in M-CSF (scale bar: 25 μm). (B) Representative flow cytometry plots of non-dband db-derived BM macrophages after 7 days culture in M-CSF, showing expression of macrophage markers CD11b and F4.80. (C) qRTPCR analysis of myeloid cell markers Emr1 (F4.80, left panel), Ly6g (Gr-1, centre panel) and Itgam (CD11b, right panel) at day 1 and day 7 of culture in M-CSF in non-db (grey bars) and db (black bars) BM macrophages from three independent RNA isolations for each condition. (D) qRT-PCR analysis of CCR2 expression in nondb (grey bar) and db (black bar) BM macrophages after 7 days of culture in M-CSF from three independent RNA isolations for each condition. (E) Chemotactic response to MCP-1 in a transwell assay showing mean number of cells per field of view (FoV) of non-db and db BM macrophages from three independent experiments. (F) Scratch wound migration assay of non-db (grey diamonds) and db (black squares) BM macrophages showing mean number of cells migrated into scratch wound at 4-hour intervals over 16 hours from three independent experiments. (G) Adhesion assay of non-db (grey bar) and db (black bar) BM macrophages showing mean number of cells per field of view adhered onto activated endothelial cells after 4 hours from three independent experiments. (H) Adhesion assays comparing classically activated (CA) and alternatively activated (AA) BM macrophages with wound-derived macrophages at days 3 and 7 following wounding ( n =3 for each condition).

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Transwell Assay, Migration, Cell Adhesion Assay

    53) Product Images from "Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice"

    Article Title: Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.012237

    Analysis of myeloid cell subsets in day-5 and day-10 non-db and db wounds. (A) Representative flow plots from tissue-dissociated single-cell suspensions of whole wounds gated on CD11b + cells and showing Gr-1 and CD14 expression in (i) isotype control, (ii) non-db day-5, (iii) db day-5, (iv) non-db day-10 and (v) db day-10 wounds. Upper left region of plot (P4) shows cells counted as positive for Gr-1, but negative for CD14. Upper right region of plot (P3) shows cells double positive for Gr-1 and CD14. Lower right region of plot (P5) shows cells negative for Gr-1 but positive for CD14. (B,C) Graphs of flow cytometry data shown in A for day-5 (B) and day-10 (C) wounds displaying percent of total wound cells positive for the listed marker combinations (grey bars, non-db; black bars, db; n =6, * P
    Figure Legend Snippet: Analysis of myeloid cell subsets in day-5 and day-10 non-db and db wounds. (A) Representative flow plots from tissue-dissociated single-cell suspensions of whole wounds gated on CD11b + cells and showing Gr-1 and CD14 expression in (i) isotype control, (ii) non-db day-5, (iii) db day-5, (iv) non-db day-10 and (v) db day-10 wounds. Upper left region of plot (P4) shows cells counted as positive for Gr-1, but negative for CD14. Upper right region of plot (P3) shows cells double positive for Gr-1 and CD14. Lower right region of plot (P5) shows cells negative for Gr-1 but positive for CD14. (B,C) Graphs of flow cytometry data shown in A for day-5 (B) and day-10 (C) wounds displaying percent of total wound cells positive for the listed marker combinations (grey bars, non-db; black bars, db; n =6, * P

    Techniques Used: Flow Cytometry, Expressing, Cytometry, Marker

    Differentiation and behavioural assays of non-db- and db-derived BM macrophages. (A) Representative bright-field images of nondb- and db-derived BM macrophages after 7 days culture in M-CSF (scale bar: 25 μm). (B) Representative flow cytometry plots of non-dband db-derived BM macrophages after 7 days culture in M-CSF, showing expression of macrophage markers CD11b and F4.80. (C) qRTPCR analysis of myeloid cell markers Emr1 (F4.80, left panel), Ly6g (Gr-1, centre panel) and Itgam (CD11b, right panel) at day 1 and day 7 of culture in M-CSF in non-db (grey bars) and db (black bars) BM macrophages from three independent RNA isolations for each condition. (D) qRT-PCR analysis of CCR2 expression in nondb (grey bar) and db (black bar) BM macrophages after 7 days of culture in M-CSF from three independent RNA isolations for each condition. (E) Chemotactic response to MCP-1 in a transwell assay showing mean number of cells per field of view (FoV) of non-db and db BM macrophages from three independent experiments. (F) Scratch wound migration assay of non-db (grey diamonds) and db (black squares) BM macrophages showing mean number of cells migrated into scratch wound at 4-hour intervals over 16 hours from three independent experiments. (G) Adhesion assay of non-db (grey bar) and db (black bar) BM macrophages showing mean number of cells per field of view adhered onto activated endothelial cells after 4 hours from three independent experiments. (H) Adhesion assays comparing classically activated (CA) and alternatively activated (AA) BM macrophages with wound-derived macrophages at days 3 and 7 following wounding ( n =3 for each condition).
    Figure Legend Snippet: Differentiation and behavioural assays of non-db- and db-derived BM macrophages. (A) Representative bright-field images of nondb- and db-derived BM macrophages after 7 days culture in M-CSF (scale bar: 25 μm). (B) Representative flow cytometry plots of non-dband db-derived BM macrophages after 7 days culture in M-CSF, showing expression of macrophage markers CD11b and F4.80. (C) qRTPCR analysis of myeloid cell markers Emr1 (F4.80, left panel), Ly6g (Gr-1, centre panel) and Itgam (CD11b, right panel) at day 1 and day 7 of culture in M-CSF in non-db (grey bars) and db (black bars) BM macrophages from three independent RNA isolations for each condition. (D) qRT-PCR analysis of CCR2 expression in nondb (grey bar) and db (black bar) BM macrophages after 7 days of culture in M-CSF from three independent RNA isolations for each condition. (E) Chemotactic response to MCP-1 in a transwell assay showing mean number of cells per field of view (FoV) of non-db and db BM macrophages from three independent experiments. (F) Scratch wound migration assay of non-db (grey diamonds) and db (black squares) BM macrophages showing mean number of cells migrated into scratch wound at 4-hour intervals over 16 hours from three independent experiments. (G) Adhesion assay of non-db (grey bar) and db (black bar) BM macrophages showing mean number of cells per field of view adhered onto activated endothelial cells after 4 hours from three independent experiments. (H) Adhesion assays comparing classically activated (CA) and alternatively activated (AA) BM macrophages with wound-derived macrophages at days 3 and 7 following wounding ( n =3 for each condition).

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Transwell Assay, Migration, Cell Adhesion Assay

    54) Product Images from "Resetting the transcription factor network reverses terminal chronic hepatic failure"

    Article Title: Resetting the transcription factor network reverses terminal chronic hepatic failure

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI73137

    Hepatocyte-enriched transcription factor network genes and liver-specific genes are severely downregulated in decompensated hepatocytes from end-stage livers. ( A ) Expression changes by qPCR in the hepatocyte transcription factor network genes Foxa2 , Hnf1a , Cebpa , and Ppara with progression from degenerative liver disease to chronic and terminal hepatic failure. ( B ) Expression of liver-specific genes and genes affected downstream of HNF4α. A1at , α1-antitrypsin; Otc, ornithine transcarbamylase; F7 , coagulation factor VII; Apoe , Apoa2 , and Apoc3 , apolipoproteins E, A2, and C3; Cyp3a23/3a1 , cytochrome P450 3a23; Tdo2 , tryptophan 2,3-dioxygenase; Tf, transferrin; Ttr , transthyretin; and Tat, tyrosine aminotransferase. qPCR was performed using three technical replicates and cDNA pooled from 4–5 animals per biological group. Each value represents the mean ± SD ( A and B ). Statistical analyses were performed using the Tukey-Kramer multiple comparisons procedure among normal hepatocytes or compensated or decompensated cirrhotic hepatocytes ( A and B , * P
    Figure Legend Snippet: Hepatocyte-enriched transcription factor network genes and liver-specific genes are severely downregulated in decompensated hepatocytes from end-stage livers. ( A ) Expression changes by qPCR in the hepatocyte transcription factor network genes Foxa2 , Hnf1a , Cebpa , and Ppara with progression from degenerative liver disease to chronic and terminal hepatic failure. ( B ) Expression of liver-specific genes and genes affected downstream of HNF4α. A1at , α1-antitrypsin; Otc, ornithine transcarbamylase; F7 , coagulation factor VII; Apoe , Apoa2 , and Apoc3 , apolipoproteins E, A2, and C3; Cyp3a23/3a1 , cytochrome P450 3a23; Tdo2 , tryptophan 2,3-dioxygenase; Tf, transferrin; Ttr , transthyretin; and Tat, tyrosine aminotransferase. qPCR was performed using three technical replicates and cDNA pooled from 4–5 animals per biological group. Each value represents the mean ± SD ( A and B ). Statistical analyses were performed using the Tukey-Kramer multiple comparisons procedure among normal hepatocytes or compensated or decompensated cirrhotic hepatocytes ( A and B , * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Coagulation

    Schematic diagram for the induction of irreversible degenerative liver disease and terminal hepatic failure in rats and changes in HNF4α expression with disease progression. ( A ) Schematic diagram of the phenobarbital and CCl 4 treatment protocol. Scale bar: 100 µm. ( B – D ) Changes in HNF4α expression with disease progression. ( B ) qPCR and ( C ) Western blot for HNF4α expression in isolated hepatocytes from livers with degenerative disease and compensated (Comp) or decompensated (Decomp) function. ( D ) Immunohistochemistry of liver tissue and isolated hepatocytes; original magnification, ×100 and (cytospins) ×200. Normal age-matched livers or hepatocytes were used as controls. β-Actin was used as the PCR and Western blot control. qPCR and Western blot analysis were performed using three technical replicates and cDNA pooled from 4–5 animals per biological group. Immunohistochemistry is representative of four images per biologic group. Each value represents the mean ± SD ( B – D ). Statistical analyses were performed using the Tukey-Kramer multiple comparisons procedure among normal hepatocytes or compensated or decompensated cirrhotic hepatocytes ( B – D , * P
    Figure Legend Snippet: Schematic diagram for the induction of irreversible degenerative liver disease and terminal hepatic failure in rats and changes in HNF4α expression with disease progression. ( A ) Schematic diagram of the phenobarbital and CCl 4 treatment protocol. Scale bar: 100 µm. ( B – D ) Changes in HNF4α expression with disease progression. ( B ) qPCR and ( C ) Western blot for HNF4α expression in isolated hepatocytes from livers with degenerative disease and compensated (Comp) or decompensated (Decomp) function. ( D ) Immunohistochemistry of liver tissue and isolated hepatocytes; original magnification, ×100 and (cytospins) ×200. Normal age-matched livers or hepatocytes were used as controls. β-Actin was used as the PCR and Western blot control. qPCR and Western blot analysis were performed using three technical replicates and cDNA pooled from 4–5 animals per biological group. Immunohistochemistry is representative of four images per biologic group. Each value represents the mean ± SD ( B – D ). Statistical analyses were performed using the Tukey-Kramer multiple comparisons procedure among normal hepatocytes or compensated or decompensated cirrhotic hepatocytes ( B – D , * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Isolation, Immunohistochemistry, Polymerase Chain Reaction

    55) Product Images from "Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice"

    Article Title: Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.012237

    Analysis of differentiation and polarisation in day-5 and day-10 wound-derived myeloid cells. (A,B) qRT-PCR analysis of (A) general macrophage maturation marker expression in wound-derived CD14 + (all) cells, or (B) general granulocyte maturation marker expression in wound-derived Gr-1 + CD14 − cells, pooled from three to six non-db or three to six db samples (three pools each) 5 days post-wounding. Bars indicate gene expression in db-derived CD14 + (all) cells relative to the non-db sample (indicated by dashed line) and reference genes Hist2h2aa1 and Hsp90ab1 . Means and s.e.m. of three biological replicates are shown (* P
    Figure Legend Snippet: Analysis of differentiation and polarisation in day-5 and day-10 wound-derived myeloid cells. (A,B) qRT-PCR analysis of (A) general macrophage maturation marker expression in wound-derived CD14 + (all) cells, or (B) general granulocyte maturation marker expression in wound-derived Gr-1 + CD14 − cells, pooled from three to six non-db or three to six db samples (three pools each) 5 days post-wounding. Bars indicate gene expression in db-derived CD14 + (all) cells relative to the non-db sample (indicated by dashed line) and reference genes Hist2h2aa1 and Hsp90ab1 . Means and s.e.m. of three biological replicates are shown (* P

    Techniques Used: Derivative Assay, Quantitative RT-PCR, Marker, Expressing

    56) Product Images from "Neuroprotective effect of placenta-derived mesenchymal stromal cells: role of exosomes"

    Article Title: Neuroprotective effect of placenta-derived mesenchymal stromal cells: role of exosomes

    Journal: The FASEB Journal

    doi: 10.1096/fj.201800972R

    Neuroprotective effect of PMSC-derived exosomes. SH-SY5Y cells were subjected to staurosporine treatment and then cultured with exosomes isolated from the conditioned medium of PMSCs. A–D ) Calcein AM staining and Wimasis image analysis in the absence ( A , B ) and presence ( C , D ) of exosomes show increased neurite outgrowth in the presence of exosomes. E–H ) WimNeuron image analysis showed a significant increase in total branching points ( E ), circuitry length ( F ), and total segments ( G ), and an increase in cell number ( H ) in the presence of exosomes compared with its control ( n = 3 donor cell banks and repeated 3 times). Scale bars, 200 µm.
    Figure Legend Snippet: Neuroprotective effect of PMSC-derived exosomes. SH-SY5Y cells were subjected to staurosporine treatment and then cultured with exosomes isolated from the conditioned medium of PMSCs. A–D ) Calcein AM staining and Wimasis image analysis in the absence ( A , B ) and presence ( C , D ) of exosomes show increased neurite outgrowth in the presence of exosomes. E–H ) WimNeuron image analysis showed a significant increase in total branching points ( E ), circuitry length ( F ), and total segments ( G ), and an increase in cell number ( H ) in the presence of exosomes compared with its control ( n = 3 donor cell banks and repeated 3 times). Scale bars, 200 µm.

    Techniques Used: Derivative Assay, Cell Culture, Isolation, Staining

    Expression and role of galectin 1 on the neuroprotective effect of PMSC exosomes. A ) Expression of galectin 1 in PMSCs (lane 1) and PMSC exosomes (lane 2) by Western blot analysis. B , C ) Flow cytometry analysis shows expression of galectin 1 on the surface of PMSCs ( B ) and PMSC exosomes ( C ) (IgG control in grey and anti-galectin 1 in red). SH-SY5Y cells were subjected to staurosporine treatment and then cultured with exosomes preincubated with either control IgG (con) or anti-galectin 1 antibody (gal1). D–F ) Calcein AM staining and subsequent Wimasis image analysis showed a significant decrease in total branching points ( D ), a decrease in total segments ( E ), and no change in circuitry length ( F ) ( n = 3 donor cell banks).
    Figure Legend Snippet: Expression and role of galectin 1 on the neuroprotective effect of PMSC exosomes. A ) Expression of galectin 1 in PMSCs (lane 1) and PMSC exosomes (lane 2) by Western blot analysis. B , C ) Flow cytometry analysis shows expression of galectin 1 on the surface of PMSCs ( B ) and PMSC exosomes ( C ) (IgG control in grey and anti-galectin 1 in red). SH-SY5Y cells were subjected to staurosporine treatment and then cultured with exosomes preincubated with either control IgG (con) or anti-galectin 1 antibody (gal1). D–F ) Calcein AM staining and subsequent Wimasis image analysis showed a significant decrease in total branching points ( D ), a decrease in total segments ( E ), and no change in circuitry length ( F ) ( n = 3 donor cell banks).

    Techniques Used: Expressing, Western Blot, Flow Cytometry, Cell Culture, Staining

    Network analysis of proteins identified in exosomes. Proteins identified by the Scaffold software were subjected to network analysis using the STRING database set at a high confidence interaction score of 0.900. Network nodes represent proteins. Colored nodes represent query proteins and first shell of interactions. Empty nodes represent proteins of unknown 3-dimensional structure. Filled nodes signify that some 3-dimensional structure is known. Edges represent protein-protein associations (proteins jointly contributing to a shared function). Known interactions are generated from curated databases and experimentally determined; predicted interactions are generated from gene neighborhood, gene fusions, and gene co-occurrence; others are generated from text mining, coexpression, and protein homology.
    Figure Legend Snippet: Network analysis of proteins identified in exosomes. Proteins identified by the Scaffold software were subjected to network analysis using the STRING database set at a high confidence interaction score of 0.900. Network nodes represent proteins. Colored nodes represent query proteins and first shell of interactions. Empty nodes represent proteins of unknown 3-dimensional structure. Filled nodes signify that some 3-dimensional structure is known. Edges represent protein-protein associations (proteins jointly contributing to a shared function). Known interactions are generated from curated databases and experimentally determined; predicted interactions are generated from gene neighborhood, gene fusions, and gene co-occurrence; others are generated from text mining, coexpression, and protein homology.

    Techniques Used: Software, Generated

    57) Product Images from "Molecular diversity and function of jasmintides from Jasminum sambac"

    Article Title: Molecular diversity and function of jasmintides from Jasminum sambac

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-018-1361-y

    Peptidomic analyses of fresh J sambac . MALDI-TOF MS profiles of ( a ) leaves, ( b ) flowers and ( c ) roots of J sambac . Zoomed MALDI-TOF MS spectra of purified ( d ) jS3 and ( e ) jS4 with monoisotopic peaks at 3199.42 and 3199.31 Da, respectively
    Figure Legend Snippet: Peptidomic analyses of fresh J sambac . MALDI-TOF MS profiles of ( a ) leaves, ( b ) flowers and ( c ) roots of J sambac . Zoomed MALDI-TOF MS spectra of purified ( d ) jS3 and ( e ) jS4 with monoisotopic peaks at 3199.42 and 3199.31 Da, respectively

    Techniques Used: Mass Spectrometry, Purification

    58) Product Images from "Neprosin, a Selective Prolyl Endoprotease for Bottom-up Proteomics and Histone Mapping *"

    Article Title: Neprosin, a Selective Prolyl Endoprotease for Bottom-up Proteomics and Histone Mapping *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M116.066803

    Cleavage specificity of neprosin. A, IceLogo visualization of neprosin preference toward substrate subsites P4–P4′, derived from HeLa lysate digestion ( n = 3316 and 3275 unique cleavage sites). Differences are displayed as the amino acid occurrence in a certain position, normalized to the natural occurrence in H. sapiens. B, heat map visualization of neprosin's cleavage specificity toward subsites P4–P4′ normalized to the natural occurrence in H. sapiens .
    Figure Legend Snippet: Cleavage specificity of neprosin. A, IceLogo visualization of neprosin preference toward substrate subsites P4–P4′, derived from HeLa lysate digestion ( n = 3316 and 3275 unique cleavage sites). Differences are displayed as the amino acid occurrence in a certain position, normalized to the natural occurrence in H. sapiens. B, heat map visualization of neprosin's cleavage specificity toward subsites P4–P4′ normalized to the natural occurrence in H. sapiens .

    Techniques Used: Derivative Assay

    Histone H3 and histone H4 mapping using neprosin as identified within a digest of whole histones from calf thymus. A, sequence coverage plot of the N terminus (amino acids 1–40) of histone H3. Underlying peptides are shown as red bars in a sequence coverage plot. The retention time of the corresponding peptides was between 3 and 8 min. Pro residues are highlighted in red and bold , and Arg and Lys residues are shown in black and bold . Only those sequences are displayed for which the PTM site was confirmed based on manual inspection. Cleavages after Pro-16, Ala-24, and Ala-25 were only observed to minor extent. Legend for identified PTMs is provided in the figure. B, sequence coverage plot of the N terminus (amino acids 1–40) of histone H4. Underlying peptides are shown as red bars . Pro residues are highlighted in red and bold , whereas Arg and Lys residues are shown in black and bold . Only those peptides are displayed for which the PTM site was confirmed based on unambiguous corresponding fragment ions. Legend for identified PTMs is provided in the figure. C, exemplary fragment ion spectrum of an acetylated and dimethylated peptide of histone H4 (residues 1–32; precursor ion was at m/z 688.6046; z = 5) showing almost complete fragment ion series using EThcD.
    Figure Legend Snippet: Histone H3 and histone H4 mapping using neprosin as identified within a digest of whole histones from calf thymus. A, sequence coverage plot of the N terminus (amino acids 1–40) of histone H3. Underlying peptides are shown as red bars in a sequence coverage plot. The retention time of the corresponding peptides was between 3 and 8 min. Pro residues are highlighted in red and bold , and Arg and Lys residues are shown in black and bold . Only those sequences are displayed for which the PTM site was confirmed based on manual inspection. Cleavages after Pro-16, Ala-24, and Ala-25 were only observed to minor extent. Legend for identified PTMs is provided in the figure. B, sequence coverage plot of the N terminus (amino acids 1–40) of histone H4. Underlying peptides are shown as red bars . Pro residues are highlighted in red and bold , whereas Arg and Lys residues are shown in black and bold . Only those peptides are displayed for which the PTM site was confirmed based on unambiguous corresponding fragment ions. Legend for identified PTMs is provided in the figure. C, exemplary fragment ion spectrum of an acetylated and dimethylated peptide of histone H4 (residues 1–32; precursor ion was at m/z 688.6046; z = 5) showing almost complete fragment ion series using EThcD.

    Techniques Used: Sequencing

    GFP S65T proteolysis by neprosin under different conditions. Neprosin activity was expressed as percentage of fluorescence lost (100% − % fluorescence recovery) relative to the untreated GFP S65T (without neprosin) under the same reaction condition. The error bars represent standard deviation from at least three replicates. A, effect of protease inhibitors and reducing agents on neprosin activity. B, effect of pH on neprosin activity. The indicated pH is the final pH of the reaction mixture. C, effect of temperature on neprosin activity. ZPP, Z-pro-prolinal inhibitor.
    Figure Legend Snippet: GFP S65T proteolysis by neprosin under different conditions. Neprosin activity was expressed as percentage of fluorescence lost (100% − % fluorescence recovery) relative to the untreated GFP S65T (without neprosin) under the same reaction condition. The error bars represent standard deviation from at least three replicates. A, effect of protease inhibitors and reducing agents on neprosin activity. B, effect of pH on neprosin activity. The indicated pH is the final pH of the reaction mixture. C, effect of temperature on neprosin activity. ZPP, Z-pro-prolinal inhibitor.

    Techniques Used: Activity Assay, Fluorescence, Standard Deviation

    Size distribution of identified proteins and peptides after HeLa whole-cell lysate digestion. A, size distribution of identified protein groups in HeLa lysate digested with neprosin ( solid red line ), LysC ( solid dark gray line ), and trypsin ( solid light gray line ) plotted against the in silico calculated size distribution of protein groups in the human proteome ( dotted black line ). B, average mass of identified peptides ( black bars ) and all MS-triggered peptide ions ( gray bars ) after digestion of HeLa cell lysate with trypsin, LysC, and neprosin as indicated.
    Figure Legend Snippet: Size distribution of identified proteins and peptides after HeLa whole-cell lysate digestion. A, size distribution of identified protein groups in HeLa lysate digested with neprosin ( solid red line ), LysC ( solid dark gray line ), and trypsin ( solid light gray line ) plotted against the in silico calculated size distribution of protein groups in the human proteome ( dotted black line ). B, average mass of identified peptides ( black bars ) and all MS-triggered peptide ions ( gray bars ) after digestion of HeLa cell lysate with trypsin, LysC, and neprosin as indicated.

    Techniques Used: In Silico, Mass Spectrometry

    Proteomic analysis of HeLa whole-cell lysate after digestion with neprosin, trypsin, and LysC. A, Venn diagram visualization of identified protein groups for HeLa lysate digested with trypsin, LysC, and neprosin. B, Venn diagram visualization of the observed sequence coverage for the 1251 proteins commonly identified from HeLa digests with trypsin, LysC, and neprosin.
    Figure Legend Snippet: Proteomic analysis of HeLa whole-cell lysate after digestion with neprosin, trypsin, and LysC. A, Venn diagram visualization of identified protein groups for HeLa lysate digested with trypsin, LysC, and neprosin. B, Venn diagram visualization of the observed sequence coverage for the 1251 proteins commonly identified from HeLa digests with trypsin, LysC, and neprosin.

    Techniques Used: Sequencing

    59) Product Images from "Developing a Novel Sulfoxide-containing MS-cleavable Homobifunctional Cysteine Reactive Cross-linker for Studying Protein-Protein Interactions"

    Article Title: Developing a Novel Sulfoxide-containing MS-cleavable Homobifunctional Cysteine Reactive Cross-linker for Studying Protein-Protein Interactions

    Journal: Analytical chemistry

    doi: 10.1021/acs.analchem.8b01287

    MS analysis of the BMSO inter-linked Ac-LR9 homodimer (α–α) (A) MS 1 spectrum of the inter-link with closed-ring form, (α c –α c ) 4+ (m/z 637.7849 4+ ). (B) MS 2 spectrum of the (α c –α c) 4+ detected in (A), in which two dominant fragment ions, i.e. α Ac (m/z 625.29 2+ ) and α Tc (m/z 641.27 2+ ), were detected as predicted for homodimer inter-links. (C) MS 1 spectrum of the inter-link with open-ring form, (α o –α o ) 4+ (m/z 646.7885 4+ ). (D) MS 2 spectrum of the (α o –α o) 4+ detected in (C), in which two dominant fragment ions, i.e. α Ao (m/z 634.29 2+ ) and α To (m/z 650.28 2+ ) were detected as expected. Note: c: closed-ring; o: open-ring; Ac/Tc: alkene/unsaturated thiol moieties with closed-ring SITE; Ao/To: alkene/unsaturated thiol moieties with open-ring SATE.
    Figure Legend Snippet: MS analysis of the BMSO inter-linked Ac-LR9 homodimer (α–α) (A) MS 1 spectrum of the inter-link with closed-ring form, (α c –α c ) 4+ (m/z 637.7849 4+ ). (B) MS 2 spectrum of the (α c –α c) 4+ detected in (A), in which two dominant fragment ions, i.e. α Ac (m/z 625.29 2+ ) and α Tc (m/z 641.27 2+ ), were detected as predicted for homodimer inter-links. (C) MS 1 spectrum of the inter-link with open-ring form, (α o –α o ) 4+ (m/z 646.7885 4+ ). (D) MS 2 spectrum of the (α o –α o) 4+ detected in (C), in which two dominant fragment ions, i.e. α Ao (m/z 634.29 2+ ) and α To (m/z 650.28 2+ ) were detected as expected. Note: c: closed-ring; o: open-ring; Ac/Tc: alkene/unsaturated thiol moieties with closed-ring SITE; Ao/To: alkene/unsaturated thiol moieties with open-ring SATE.

    Techniques Used: Mass Spectrometry

    60) Product Images from "The nature and extent of contributions by defective ribosome products to the HLA peptidome"

    Article Title: The nature and extent of contributions by defective ribosome products to the HLA peptidome

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

    doi: 10.1073/pnas.1321902111

    Relative H/L ratios of HLA peptides and of their source proteins at the 12-h time points of the JY ( A ), U937 ( B ), and RPMI8226 ( C ) cells. The different subunits of ribosomes are labeled as blue circles and the subunits of TCP-1 as red squares.
    Figure Legend Snippet: Relative H/L ratios of HLA peptides and of their source proteins at the 12-h time points of the JY ( A ), U937 ( B ), and RPMI8226 ( C ) cells. The different subunits of ribosomes are labeled as blue circles and the subunits of TCP-1 as red squares.

    Techniques Used: Labeling

    61) Product Images from "A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens *"

    Article Title: A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M116.063800

    Sequence analysis of the phosphorylated and the nonphosphorylated peptides associated to HLA-B*39 ( left column ), HLA-B*27 ( middle column ) or HLA-B*07 ( right column ). . A , Frequency distribution of phosphorylation at each peptide position in the phospholigandomes bound to these allotypes, revealing the preference for phosphorylation at P4. B and C , Sequence logos of the phosphopeptides ( B ) and the nonmodified peptides ( C ) identified in the ligandomes of these HLA-B molecules.
    Figure Legend Snippet: Sequence analysis of the phosphorylated and the nonphosphorylated peptides associated to HLA-B*39 ( left column ), HLA-B*27 ( middle column ) or HLA-B*07 ( right column ). . A , Frequency distribution of phosphorylation at each peptide position in the phospholigandomes bound to these allotypes, revealing the preference for phosphorylation at P4. B and C , Sequence logos of the phosphopeptides ( B ) and the nonmodified peptides ( C ) identified in the ligandomes of these HLA-B molecules.

    Techniques Used: Sequencing

    Frequency distribution of phosphorylation among the identified HLA-B*40 phosphopeptides ( blue ) and the in silico predicted HLA-B*40 binders ( red ).
    Figure Legend Snippet: Frequency distribution of phosphorylation among the identified HLA-B*40 phosphopeptides ( blue ) and the in silico predicted HLA-B*40 binders ( red ).

    Techniques Used: In Silico

    62) Product Images from "ELTA: Enzymatic Labeling of Terminal ADP-ribose"

    Article Title: ELTA: Enzymatic Labeling of Terminal ADP-ribose

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.12.022

    ELTA labels free or protein-conjugated ADP-ribose monomers and polymers. (a) Schematics of ELTA. Free or protein-conjugated ADP-ribose can be labeled by incubating with OAS1 and dATP, where the 2’-OH terminus is indicated in red. Colored box indicates various dATP analogs that can also be used in the ELTA reactions, including radioactive ( 32 P), fluorescent (Cy3, Cy5), biotinylated or clickable analogs. (b) 15% urea-PAGE analyses of the addition of 32 P-dAMP onto ADP-ribose monomers and polymers using ELTA and visualized by autoradiograph. (c) MALDI-TOF analyses of the reaction of ADP-ribose with dATP, and with or without OAS1. (d-e) Analyses of the ELTA labeling reaction of (d) MARylated PARP10 catalytic domain (mod-PARP10 cd ) and (e) PARylated ha PARP (mod- ha PARP) using 32 P-dATP. Shown are a coomassie gel (left), an autoradiograph (middle), and a western blot probed with pan-ADP-ribose reagent (right). As negative controls, modified proteins were treated with the phosphodiesterase hs NudT16 to remove the 2’-OH termini of the ADP-ribose groups prior to ELTA labeling. For panel d, * indicates PARP10; OAS1 was ADP-ribosylated by PARP10 with the remnant of NAD + , and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1. For panel e, * indicates ha PARP and § indicates ha PARP fragments that were also ADP-ribosylated and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1.
    Figure Legend Snippet: ELTA labels free or protein-conjugated ADP-ribose monomers and polymers. (a) Schematics of ELTA. Free or protein-conjugated ADP-ribose can be labeled by incubating with OAS1 and dATP, where the 2’-OH terminus is indicated in red. Colored box indicates various dATP analogs that can also be used in the ELTA reactions, including radioactive ( 32 P), fluorescent (Cy3, Cy5), biotinylated or clickable analogs. (b) 15% urea-PAGE analyses of the addition of 32 P-dAMP onto ADP-ribose monomers and polymers using ELTA and visualized by autoradiograph. (c) MALDI-TOF analyses of the reaction of ADP-ribose with dATP, and with or without OAS1. (d-e) Analyses of the ELTA labeling reaction of (d) MARylated PARP10 catalytic domain (mod-PARP10 cd ) and (e) PARylated ha PARP (mod- ha PARP) using 32 P-dATP. Shown are a coomassie gel (left), an autoradiograph (middle), and a western blot probed with pan-ADP-ribose reagent (right). As negative controls, modified proteins were treated with the phosphodiesterase hs NudT16 to remove the 2’-OH termini of the ADP-ribose groups prior to ELTA labeling. For panel d, * indicates PARP10; OAS1 was ADP-ribosylated by PARP10 with the remnant of NAD + , and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1. For panel e, * indicates ha PARP and § indicates ha PARP fragments that were also ADP-ribosylated and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Western Blot, Modification

    63) Product Images from "Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice"

    Article Title: Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.012237

    Analysis of Arg1 + cells in human healing and non-healing diabetic foot ulcers. (A) Cartoon diagram showing punch biopsy site, indicated by blue dotted circle. (B) H E stain of whole-wound section showing representative areas used for analyses of CD68, Arg1 and Nos2 immunofluorescent staining (scale bar: 600 μm). (C) Representative images of CD68 and Arg1 immunofluorescent staining in sections from healing and non-healing diabetic foot ulcers. The first three panels of each group show fluorescent staining as indicated (scale bar: 100 μm). The fourth panel of each group shows a high-magnification view of the area indicated by the white box in the merge panels (scale bar: 25 μm). (D) Quantification of the number of CD68 + cells per mm 2 in healing and non-healing wounds. (E) Quantification of the percentage of macrophages (Macs) that are Arg1 + (double positive for Arg1 and CD68) in healing and non-healing wounds ( n =7, * P
    Figure Legend Snippet: Analysis of Arg1 + cells in human healing and non-healing diabetic foot ulcers. (A) Cartoon diagram showing punch biopsy site, indicated by blue dotted circle. (B) H E stain of whole-wound section showing representative areas used for analyses of CD68, Arg1 and Nos2 immunofluorescent staining (scale bar: 600 μm). (C) Representative images of CD68 and Arg1 immunofluorescent staining in sections from healing and non-healing diabetic foot ulcers. The first three panels of each group show fluorescent staining as indicated (scale bar: 100 μm). The fourth panel of each group shows a high-magnification view of the area indicated by the white box in the merge panels (scale bar: 25 μm). (D) Quantification of the number of CD68 + cells per mm 2 in healing and non-healing wounds. (E) Quantification of the percentage of macrophages (Macs) that are Arg1 + (double positive for Arg1 and CD68) in healing and non-healing wounds ( n =7, * P

    Techniques Used: Staining, Magnetic Cell Separation

    Analysis of inflammatory cell polarisation in wounds of non-diabetic and diabetic mice over a healing time-course . (A) Overview of a representative whole-wound section from a day-7 diabetic wound with boxes indicating where images were captured for analyses (scale bar: 1 mm). (B) Immunofluorescent detection of CD45, Nos2 and Arg1 in day-4 (i) and day-7 (ii) wounds of non-db and db mice. Merged images show individual CD45 + cells that are also positive for Nos2 but not Arg1 (M1 phenotype, yellow arrow), Arg1 but not Nos2 (M2 phenotype, blue arrow), and for both Nos2 and Arg1 (‘mixed’ phenotype, white arrows) (scale bar: 10 μm). (C,D) Quantification of macrophage phenotypes as shown in B at (C) day 4 following wounding and (D) day 7 following wounding, from six non-db and six db mice at each time point. Non-db, non-diabetic; db, diabetic; ** P
    Figure Legend Snippet: Analysis of inflammatory cell polarisation in wounds of non-diabetic and diabetic mice over a healing time-course . (A) Overview of a representative whole-wound section from a day-7 diabetic wound with boxes indicating where images were captured for analyses (scale bar: 1 mm). (B) Immunofluorescent detection of CD45, Nos2 and Arg1 in day-4 (i) and day-7 (ii) wounds of non-db and db mice. Merged images show individual CD45 + cells that are also positive for Nos2 but not Arg1 (M1 phenotype, yellow arrow), Arg1 but not Nos2 (M2 phenotype, blue arrow), and for both Nos2 and Arg1 (‘mixed’ phenotype, white arrows) (scale bar: 10 μm). (C,D) Quantification of macrophage phenotypes as shown in B at (C) day 4 following wounding and (D) day 7 following wounding, from six non-db and six db mice at each time point. Non-db, non-diabetic; db, diabetic; ** P

    Techniques Used: Mouse Assay

    64) Product Images from "Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells"

    Article Title: Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075517

    miR-200b eRNA is transcribed from the upstream intergenic enhancer region of miR-200b~200a~429. (A) Schematic representation of the miR-200b~200a~429 locus. Black box indicates the position of the potential enhancer. A black arrow marks the TSS direction of the primary miR-200b~200a~429 transcript. Grey boxes indicated the mature miR-200b, miR-200a and miR-429 genes. Black bars indicate the positions (in kilobases, kb) of the PCR primers used for qRT-PCR. (B) Expression levels of HOTAIR, miR-200b eRNA and the primary miR-200b~200a~429 transcript as determined by qRT-PCR in epithelial and mesenchymal HMLE cells using random hexamer primed cDNA synthesized from total RNA. The x-axis shows the distance from the miR-200b~200a~429 TSS in kb. Data represents mean ± SD of three independent experiments. (C) Schematic representation of the enhancer region located relative to the miR-200b~200a~429 TSS. Boxes indicate the locations of PCR amplicons used to detect the miR-200b eRNA in Figure 3B . RACE PCR primers and their start locations relative to the miR-200b~200a~429 are indicated. 5’ and 3’ RACE-seq analysis of the miR-200b eRNA with cDNA prepared from total RNA of HMLE, mesHMLE, MDA-MB-468 and MDA-MB-231 cells as described in the Materials and Methods. 5’ and 3’ ends of the miR-200b eRNA transcript are mapped as % total reads for each cell line with extreme 5’ and 3’ ends indicated by colored arrows below. A consensus miR-200b eRNA transcript is indicated.
    Figure Legend Snippet: miR-200b eRNA is transcribed from the upstream intergenic enhancer region of miR-200b~200a~429. (A) Schematic representation of the miR-200b~200a~429 locus. Black box indicates the position of the potential enhancer. A black arrow marks the TSS direction of the primary miR-200b~200a~429 transcript. Grey boxes indicated the mature miR-200b, miR-200a and miR-429 genes. Black bars indicate the positions (in kilobases, kb) of the PCR primers used for qRT-PCR. (B) Expression levels of HOTAIR, miR-200b eRNA and the primary miR-200b~200a~429 transcript as determined by qRT-PCR in epithelial and mesenchymal HMLE cells using random hexamer primed cDNA synthesized from total RNA. The x-axis shows the distance from the miR-200b~200a~429 TSS in kb. Data represents mean ± SD of three independent experiments. (C) Schematic representation of the enhancer region located relative to the miR-200b~200a~429 TSS. Boxes indicate the locations of PCR amplicons used to detect the miR-200b eRNA in Figure 3B . RACE PCR primers and their start locations relative to the miR-200b~200a~429 are indicated. 5’ and 3’ RACE-seq analysis of the miR-200b eRNA with cDNA prepared from total RNA of HMLE, mesHMLE, MDA-MB-468 and MDA-MB-231 cells as described in the Materials and Methods. 5’ and 3’ ends of the miR-200b eRNA transcript are mapped as % total reads for each cell line with extreme 5’ and 3’ ends indicated by colored arrows below. A consensus miR-200b eRNA transcript is indicated.

    Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Random Hexamer Labeling, Synthesized, Multiple Displacement Amplification

    65) Product Images from "Data set of Aspergillus flavus induced alterations in tear proteome: Understanding the pathogen-induced host response to fungal infection"

    Article Title: Data set of Aspergillus flavus induced alterations in tear proteome: Understanding the pathogen-induced host response to fungal infection

    Journal: Data in Brief

    doi: 10.1016/j.dib.2016.11.003

    Workflow followed for protein identification using proteome discoverer. Raw files generated by the Orbitrap Velos Pro mass spectrometer were searched against the Human protein sequences from UniProt using two different search algorithms. The PSMs from each search result was subsequently validated by the Percolator algorithm.
    Figure Legend Snippet: Workflow followed for protein identification using proteome discoverer. Raw files generated by the Orbitrap Velos Pro mass spectrometer were searched against the Human protein sequences from UniProt using two different search algorithms. The PSMs from each search result was subsequently validated by the Percolator algorithm.

    Techniques Used: Generated, Mass Spectrometry

    66) Product Images from "Dynamin-Like Proteins of Endocytosis in Plants Are Coopted by Potyviruses To Enhance Virus Infection"

    Article Title: Dynamin-Like Proteins of Endocytosis in Plants Are Coopted by Potyviruses To Enhance Virus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.01320-18

    A dynamin-like protein in soybean is associated with Soybean mosaic virus virions mediated by SMV capsid components. (A) Analysis of SMV virions purified from infected soybean leaves. The SMV preparation was separated by 10% SDS-PAGE and stained with Coomassie brilliant blue solution. Five individual bands are indicated with arrowheads; they were excised from the gel, digested with trypsin, and analyzed by LC-MS/MS. (B) Protein-protein interaction assay between GmSDL5A and SMV proteins by using a membrane yeast two-hybrid method. Control (+) yeast cells were cotransformed with TuMV 6K2 and AtVAP27-1. (C) BiFC assay of interactions between GmSDL5A and SMV VPg, CP, and CI in N. benthamiana cells. The YFP field, FM4-64 dye staining, and overlay of them are shown. Split YFP halves (YN and YC) were fused to the C termini. White arrows indicate the colocalization of the interaction complex of GmSDL5A and SMV proteins, with the endosome compartments labeled by FM4-64. Scale bar = 20 μm. (D) BiFC assay of GmSDL5A and SMV VPg, CP, and CI in N. benthamiana cells. YC and YN were exchanged for each pair tested in panel C. Representative negative controls are given. Scale bar = 20 μm.
    Figure Legend Snippet: A dynamin-like protein in soybean is associated with Soybean mosaic virus virions mediated by SMV capsid components. (A) Analysis of SMV virions purified from infected soybean leaves. The SMV preparation was separated by 10% SDS-PAGE and stained with Coomassie brilliant blue solution. Five individual bands are indicated with arrowheads; they were excised from the gel, digested with trypsin, and analyzed by LC-MS/MS. (B) Protein-protein interaction assay between GmSDL5A and SMV proteins by using a membrane yeast two-hybrid method. Control (+) yeast cells were cotransformed with TuMV 6K2 and AtVAP27-1. (C) BiFC assay of interactions between GmSDL5A and SMV VPg, CP, and CI in N. benthamiana cells. The YFP field, FM4-64 dye staining, and overlay of them are shown. Split YFP halves (YN and YC) were fused to the C termini. White arrows indicate the colocalization of the interaction complex of GmSDL5A and SMV proteins, with the endosome compartments labeled by FM4-64. Scale bar = 20 μm. (D) BiFC assay of GmSDL5A and SMV VPg, CP, and CI in N. benthamiana cells. YC and YN were exchanged for each pair tested in panel C. Representative negative controls are given. Scale bar = 20 μm.

    Techniques Used: Purification, Infection, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Protein Protein Interaction Assay, Bimolecular Fluorescence Complementation Assay, Labeling

    67) Product Images from "Preformulation and stability in biological fluids of the retrocyclin RC-101, a potential anti-HIV topical microbicide"

    Article Title: Preformulation and stability in biological fluids of the retrocyclin RC-101, a potential anti-HIV topical microbicide

    Journal: AIDS Research and Therapy

    doi: 10.1186/1742-6405-8-27

    Effect of pH on RC-101 . A) RC-101 under different pH conditions analyzed over time by HPLC. (open square) pH 3, (solid circle) pH 4, (open triangle) pH 7, and (solid square) pH 12. B) CD spectra of RC-101 solution (500 μg/mL) under different pH conditions.
    Figure Legend Snippet: Effect of pH on RC-101 . A) RC-101 under different pH conditions analyzed over time by HPLC. (open square) pH 3, (solid circle) pH 4, (open triangle) pH 7, and (solid square) pH 12. B) CD spectra of RC-101 solution (500 μg/mL) under different pH conditions.

    Techniques Used: High Performance Liquid Chromatography

    Effects of temperature on RC-101 (500 μg/mL) solutions . A) HPLC analysis for RC-101 stored at (solid circle) 25 ± C, (open square) 37 ± C, and (solid triangle) 65 ± C. B)MALDI-TOF MS spectrum of RC-101 in water, exposed for 10 days at room temperature, 100% intensity = 38291 counts.
    Figure Legend Snippet: Effects of temperature on RC-101 (500 μg/mL) solutions . A) HPLC analysis for RC-101 stored at (solid circle) 25 ± C, (open square) 37 ± C, and (solid triangle) 65 ± C. B)MALDI-TOF MS spectrum of RC-101 in water, exposed for 10 days at room temperature, 100% intensity = 38291 counts.

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry

    Effect of hydrogen peroxide on RC-101 . A) RC-101 under different concentrations of hydrogen peroxide over time analyzed by HPLC. B) RC-101 exposed to hydrogen peroxide 0.002% without EDTA (solid circle), and in the presence of EDTA (open square), over time, analyzed by HPLC.
    Figure Legend Snippet: Effect of hydrogen peroxide on RC-101 . A) RC-101 under different concentrations of hydrogen peroxide over time analyzed by HPLC. B) RC-101 exposed to hydrogen peroxide 0.002% without EDTA (solid circle), and in the presence of EDTA (open square), over time, analyzed by HPLC.

    Techniques Used: High Performance Liquid Chromatography

    Representative LC-MS/MS chromatograms for A) Sample A supernatant (blank HVF) at time 0, B) Sample B supernatant (RC-101 solution + HVF) at 72 h, C) Sample C supernatant (RC-101 solution control), and D) Sample D (RC-101 film + HVF) supernatant at 48 h .
    Figure Legend Snippet: Representative LC-MS/MS chromatograms for A) Sample A supernatant (blank HVF) at time 0, B) Sample B supernatant (RC-101 solution + HVF) at 72 h, C) Sample C supernatant (RC-101 solution control), and D) Sample D (RC-101 film + HVF) supernatant at 48 h .

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    68) Product Images from "Antitumor effect of antitissue factor antibody‐MMAE conjugate in human pancreatic tumor xenografts"

    Article Title: Antitumor effect of antitissue factor antibody‐MMAE conjugate in human pancreatic tumor xenografts

    Journal: International Journal of Cancer

    doi: 10.1002/ijc.29492

    Preparation and characterization of ADCs. ( a ) Structure of ADC. Approximately three molecules of MMAE were conjugated to one antibody by a valine‐citrulline dipeptide linker. ( b ) Particle size of mAbs and ADCs. The mean particle sizes of mAbs were 9.5 nm (anti‐human mAb) and 7.6 nm (anti‐mouse mAb) and the mean particle sizes of ADCs were 12.0 nm (anti‐human ADC) and 12.6 nm (anti‐mouse ADC). ( c ) SDS‐PAGE of mAbs and ADCs. M: molecular markers. D) Releasing of MMAE from ADCs. The rates of free MMAE released from the anti‐human and ‐mouse ADCs in an acidic buffer with cathepsin B at 37 °C for 24 hr was 72.2% and 70.8%, respectively. None of the free MMAE was released from both the anti‐human and ‐mouse ADCs in a neutral buffer without cathepsin B at 37 °C for up to 48 hr.
    Figure Legend Snippet: Preparation and characterization of ADCs. ( a ) Structure of ADC. Approximately three molecules of MMAE were conjugated to one antibody by a valine‐citrulline dipeptide linker. ( b ) Particle size of mAbs and ADCs. The mean particle sizes of mAbs were 9.5 nm (anti‐human mAb) and 7.6 nm (anti‐mouse mAb) and the mean particle sizes of ADCs were 12.0 nm (anti‐human ADC) and 12.6 nm (anti‐mouse ADC). ( c ) SDS‐PAGE of mAbs and ADCs. M: molecular markers. D) Releasing of MMAE from ADCs. The rates of free MMAE released from the anti‐human and ‐mouse ADCs in an acidic buffer with cathepsin B at 37 °C for 24 hr was 72.2% and 70.8%, respectively. None of the free MMAE was released from both the anti‐human and ‐mouse ADCs in a neutral buffer without cathepsin B at 37 °C for up to 48 hr.

    Techniques Used: SDS Page

    Affinity and Internalization of ADCs. ( a ) TF expression in the four human pancreatic cancer cell lines. Relative TF expression in BxPC‐3, PSN‐1, Capan‐1 and Panc‐1, normalized by a negative control, were 244.2, 45.2, 7.8 and 3.7, respectively, thus showing that BxPC‐3 cells had a high TF expression, PSN‐1 had a moderate TF expression and Capan‐1 and Panc‐1 had low TF expression. ( b ) Affinity of mAbs and ADCs to pancreatic cancer cells. Anti‐human mAb and anti‐human ADC were reacted to BxPC‐3 cells. Meanwhile anti‐mouse mAb, anti‐mouse ADC, control mAb and control ADC did not recognize human cells. ( c ) Internalization of ADC. The anti‐human ADC was sufficiently internalized into the cytoplasm after a 3‐hr incubation at 37°C and localized in a lysosome. Anti‐human ADC and lysosomes were indicated red and green, respectively. Colocalization of anti‐human ADC and lysosomes was merged as yellow. Scale bar represents 10 μm.
    Figure Legend Snippet: Affinity and Internalization of ADCs. ( a ) TF expression in the four human pancreatic cancer cell lines. Relative TF expression in BxPC‐3, PSN‐1, Capan‐1 and Panc‐1, normalized by a negative control, were 244.2, 45.2, 7.8 and 3.7, respectively, thus showing that BxPC‐3 cells had a high TF expression, PSN‐1 had a moderate TF expression and Capan‐1 and Panc‐1 had low TF expression. ( b ) Affinity of mAbs and ADCs to pancreatic cancer cells. Anti‐human mAb and anti‐human ADC were reacted to BxPC‐3 cells. Meanwhile anti‐mouse mAb, anti‐mouse ADC, control mAb and control ADC did not recognize human cells. ( c ) Internalization of ADC. The anti‐human ADC was sufficiently internalized into the cytoplasm after a 3‐hr incubation at 37°C and localized in a lysosome. Anti‐human ADC and lysosomes were indicated red and green, respectively. Colocalization of anti‐human ADC and lysosomes was merged as yellow. Scale bar represents 10 μm.

    Techniques Used: Expressing, Negative Control, Incubation

    69) Product Images from "PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex"

    Article Title: PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06665-5

    PWWP2A interacts with members of a core NuRD complex via MTA1. a Volcano plot of label-free interaction of GFP–PWWP2A-associated mononucleosomes. Significantly enriched proteins over GFP-associated mononucleosomes are shown in the upper right part. t test differences were obtained by two-sample t test. PWWP2A is highlighted in green, members of the core NuRD (M1HR) complex in red, previously identified H2A.Z-mononucleosome binders 12 in blue, PWWP2A-specific interactors not found in H2A.Z pulldowns 12 in black and background binding proteins in gray. b Immunoblots of several NuRD members (MTA1, HDAC2, RBBP7, RBBP4, and CHD4) and H3 upon GST and GST–PWWP2A IP with HK cell-derived mononucleosomes. c Upper part: schematic depiction of mammalian MTA1-3 paralogues. Lower part: immunoblots of PWWP2A or MBD3 after IP of endogenously tagged MTA1–FLAG, MTA2–GFP, or MTA3–FLAG from mouse embryonic stem cell (mESC) nuclear extracts. Input lanes represent 10% of the lysate used for the IP. d FLAG-PWWP2A IPs with cell lysates from HEK293 cells co-transfected with combinations of plasmids encoding FLAG-PWWP2A, HDAC1 (tagless), HA-RBBP4, and either HA-MTA1 or HA-MTA2. Left panel: western blot of inputs. Right panel: SYPRO Ruby-stained SDS-PAGE of the precipitated proteins. e Top: schematic depiction of domain structure of PWWP2A and deletion constructs. Bottom: coomassie-stained SDS–PAGE gel with indicated recombinant PWWP2A deletion constructs on beads (left) and immunoblots of IPs from lysates from HEK293 cells expressing HA-MTA1 (right)
    Figure Legend Snippet: PWWP2A interacts with members of a core NuRD complex via MTA1. a Volcano plot of label-free interaction of GFP–PWWP2A-associated mononucleosomes. Significantly enriched proteins over GFP-associated mononucleosomes are shown in the upper right part. t test differences were obtained by two-sample t test. PWWP2A is highlighted in green, members of the core NuRD (M1HR) complex in red, previously identified H2A.Z-mononucleosome binders 12 in blue, PWWP2A-specific interactors not found in H2A.Z pulldowns 12 in black and background binding proteins in gray. b Immunoblots of several NuRD members (MTA1, HDAC2, RBBP7, RBBP4, and CHD4) and H3 upon GST and GST–PWWP2A IP with HK cell-derived mononucleosomes. c Upper part: schematic depiction of mammalian MTA1-3 paralogues. Lower part: immunoblots of PWWP2A or MBD3 after IP of endogenously tagged MTA1–FLAG, MTA2–GFP, or MTA3–FLAG from mouse embryonic stem cell (mESC) nuclear extracts. Input lanes represent 10% of the lysate used for the IP. d FLAG-PWWP2A IPs with cell lysates from HEK293 cells co-transfected with combinations of plasmids encoding FLAG-PWWP2A, HDAC1 (tagless), HA-RBBP4, and either HA-MTA1 or HA-MTA2. Left panel: western blot of inputs. Right panel: SYPRO Ruby-stained SDS-PAGE of the precipitated proteins. e Top: schematic depiction of domain structure of PWWP2A and deletion constructs. Bottom: coomassie-stained SDS–PAGE gel with indicated recombinant PWWP2A deletion constructs on beads (left) and immunoblots of IPs from lysates from HEK293 cells expressing HA-MTA1 (right)

    Techniques Used: Binding Assay, Western Blot, Derivative Assay, Transfection, Staining, SDS Page, Construct, Recombinant, Expressing

    IC distinguishes between H2A and H2A.Z, whereas IN recognizes nucleosomal linker DNA. a Schematic representation of recombinant GST–PWWP2A deletions (GST-I, GST–IN, and GST-IC) used in cEMSAs. b Representative cEMSA in which a 1:1 mixture of H2A- and H2A.Z-containing nucleosomes (each with a distinct fluorescent tag) was incubated with increasing concentrations of GST-tagged I-domain constructs. The top gel shows detection of the H2A.Z nucleosomes and the bottom gel detection of the H2A nucleosomes. In both cases, the DNA contained a 20-bp linker DNA (Widom 601-sequence) on each side of the nucleosome (20–Θ–20). GST alone served as negative control. * Free DNA, ** nucleosome, *** nucleosome GST–protein complex. Arrow indicates loss of signal when nucleosome GST–protein complexes are formed. c Left: representative cEMSAs similar to ( b ) using recombinant wildtype mononucleosomes (WT, top) or mononucleosomes lacking single histone tails (TL, bottom) containing 20-bp linker DNA (20–Θ–20). Nucleosomes were incubated with the indicated concentrations of GST–IN and the gel visualized by fluorescence detection of the indicated nucleosome. Right: quantification of signal intensities of nucleosomes (**) using Image Studio Lite Ver 5.2 (LI-COR). Error bars indicate SEM of three independent replicates. d Representative EMSA using Cy-5 labeled 187-bp dsDNA and the indicated concentrations of GST–IN and GST-IC. * free DNA, *** DNA–GST–protein complex. Arrow indicates unbound DNA. e Left: representative cEMSAs similar to ( b ) using recombinant H2A.Z-containing mononucleosomes without (0–Θ–0, top) and with (20–Θ–20, bottom) linker DNA; these nucleosomes were incubated with the indicated concentrations of GST-I, GST–IN, and GST-IC. * Free DNA, ** nucleosome, *** nucleosome GST–protein complex. Arrow indicates loss of signal when nucleosome GST–protein complexes are formed. Right: Quantification of signal intensities of nucleosomes ( ** ) using Image Studio Lite Ver 5.2 (LI-COR). Error bars indicate SEM of three independent replicates
    Figure Legend Snippet: IC distinguishes between H2A and H2A.Z, whereas IN recognizes nucleosomal linker DNA. a Schematic representation of recombinant GST–PWWP2A deletions (GST-I, GST–IN, and GST-IC) used in cEMSAs. b Representative cEMSA in which a 1:1 mixture of H2A- and H2A.Z-containing nucleosomes (each with a distinct fluorescent tag) was incubated with increasing concentrations of GST-tagged I-domain constructs. The top gel shows detection of the H2A.Z nucleosomes and the bottom gel detection of the H2A nucleosomes. In both cases, the DNA contained a 20-bp linker DNA (Widom 601-sequence) on each side of the nucleosome (20–Θ–20). GST alone served as negative control. * Free DNA, ** nucleosome, *** nucleosome GST–protein complex. Arrow indicates loss of signal when nucleosome GST–protein complexes are formed. c Left: representative cEMSAs similar to ( b ) using recombinant wildtype mononucleosomes (WT, top) or mononucleosomes lacking single histone tails (TL, bottom) containing 20-bp linker DNA (20–Θ–20). Nucleosomes were incubated with the indicated concentrations of GST–IN and the gel visualized by fluorescence detection of the indicated nucleosome. Right: quantification of signal intensities of nucleosomes (**) using Image Studio Lite Ver 5.2 (LI-COR). Error bars indicate SEM of three independent replicates. d Representative EMSA using Cy-5 labeled 187-bp dsDNA and the indicated concentrations of GST–IN and GST-IC. * free DNA, *** DNA–GST–protein complex. Arrow indicates unbound DNA. e Left: representative cEMSAs similar to ( b ) using recombinant H2A.Z-containing mononucleosomes without (0–Θ–0, top) and with (20–Θ–20, bottom) linker DNA; these nucleosomes were incubated with the indicated concentrations of GST-I, GST–IN, and GST-IC. * Free DNA, ** nucleosome, *** nucleosome GST–protein complex. Arrow indicates loss of signal when nucleosome GST–protein complexes are formed. Right: Quantification of signal intensities of nucleosomes ( ** ) using Image Studio Lite Ver 5.2 (LI-COR). Error bars indicate SEM of three independent replicates

    Techniques Used: Recombinant, Incubation, Construct, Sequencing, Negative Control, Fluorescence, Labeling

    PWWP domain binds nucleic acids and S_PWWP interacts with H3K36me3. a Representative cEMSA using recombinant H2A.Z-containing mononucleosomes assembled either without (0–Θ–0, top) or with linker DNA (20–Θ–20, bottom) incubated with indicated increasing concentrations of GST–PWWP. GST alone served as negative control. * Free DNA, ** nucleosome, *** nucleosome GST–PWWP complex. Arrow indicates loss of signal when nucleosome GST–protein complexes are formed. b In silico structure of PWWP domain modeled with the web browser-based tool iTASSER and visualized with Chimera (1.8.0). β-barrels (β1–β5) are colored in red, α-helixes (α1–α3), and η-helix in blue and the three residues forming the aromatic cage (F666, W669, and W695) are highlighted in green and depicted in stick mode. NT = N-terminus, CT = C-terminus. c Top left: schematic representation of recombinant GST–PWWP2A and deletions (GST-P1, GST-I, GST-I_S, GST-I_S_PWWP, GST-S_PWWP, and GST–PWWP) used in cell-derived mononucleosome-IPs. Top right: Immunoblotting of different histone PTMs upon GST–PWWP2A deletion construct (GST–PWWP2A, GST-P1, GST-I, GST-I_S, GST-I_S_PWWP, GST-S_PWWP, and GST–PWWP) IPs with HK cell-derived mononucleosomes. Notice enrichment of H3K36me3 in comparison to other modifications in S_PWWP pulldown. GST alone served as negative control. Bottom: Data quantification was done for three biological replicates for each PTM ( n = 3). Data shown are means and error bars depict SEM. d Left: immunoblotting of H3K36me3 and H2A.Z upon GST–PWWP2A, GST-PWWP2A_ΔIC and GST-S_PWWP IPs with HK cell-derived mononucleosomes. Right: Data quantification of H3K36me3 enrichment (middle) and H2A.Z binding (right) was done for three biological replicates ( n = 3). Data shown are means and error bars depict SEM. e Left: immunoblotting of H3K36me3 upon GST-S_PWWP aromatic cage point mutants (GST-S_PWWP_F666A, GST-S_PWWP_W669A, GST-S_PWWP_W695A) IPs with HK cell-derived mononucleosomes. Notice reduction of H3K36me3 in GST-S_PWWP_W669A and GST-S_PWWP_W695A pulldowns. Right: Data quantification was done for three biological replicates ( n = 3). Data shown are means and error bars depict SEM
    Figure Legend Snippet: PWWP domain binds nucleic acids and S_PWWP interacts with H3K36me3. a Representative cEMSA using recombinant H2A.Z-containing mononucleosomes assembled either without (0–Θ–0, top) or with linker DNA (20–Θ–20, bottom) incubated with indicated increasing concentrations of GST–PWWP. GST alone served as negative control. * Free DNA, ** nucleosome, *** nucleosome GST–PWWP complex. Arrow indicates loss of signal when nucleosome GST–protein complexes are formed. b In silico structure of PWWP domain modeled with the web browser-based tool iTASSER and visualized with Chimera (1.8.0). β-barrels (β1–β5) are colored in red, α-helixes (α1–α3), and η-helix in blue and the three residues forming the aromatic cage (F666, W669, and W695) are highlighted in green and depicted in stick mode. NT = N-terminus, CT = C-terminus. c Top left: schematic representation of recombinant GST–PWWP2A and deletions (GST-P1, GST-I, GST-I_S, GST-I_S_PWWP, GST-S_PWWP, and GST–PWWP) used in cell-derived mononucleosome-IPs. Top right: Immunoblotting of different histone PTMs upon GST–PWWP2A deletion construct (GST–PWWP2A, GST-P1, GST-I, GST-I_S, GST-I_S_PWWP, GST-S_PWWP, and GST–PWWP) IPs with HK cell-derived mononucleosomes. Notice enrichment of H3K36me3 in comparison to other modifications in S_PWWP pulldown. GST alone served as negative control. Bottom: Data quantification was done for three biological replicates for each PTM ( n = 3). Data shown are means and error bars depict SEM. d Left: immunoblotting of H3K36me3 and H2A.Z upon GST–PWWP2A, GST-PWWP2A_ΔIC and GST-S_PWWP IPs with HK cell-derived mononucleosomes. Right: Data quantification of H3K36me3 enrichment (middle) and H2A.Z binding (right) was done for three biological replicates ( n = 3). Data shown are means and error bars depict SEM. e Left: immunoblotting of H3K36me3 upon GST-S_PWWP aromatic cage point mutants (GST-S_PWWP_F666A, GST-S_PWWP_W669A, GST-S_PWWP_W695A) IPs with HK cell-derived mononucleosomes. Notice reduction of H3K36me3 in GST-S_PWWP_W669A and GST-S_PWWP_W695A pulldowns. Right: Data quantification was done for three biological replicates ( n = 3). Data shown are means and error bars depict SEM

    Techniques Used: Recombinant, Incubation, Negative Control, In Silico, Derivative Assay, Construct, Binding Assay

    70) Product Images from "Dichlorinated and Brominated Rugulovasines, Ergot Alkaloids Produced by Talaromyces wortmannii"

    Article Title: Dichlorinated and Brominated Rugulovasines, Ergot Alkaloids Produced by Talaromyces wortmannii

    Journal: Molecules

    doi: 10.3390/molecules200917627

    BPC chromatogram (100–1000 Da) from the micro-extract of T. wortmannii growth in PDA medium. Compounds identified by UV/Vis-HRMS based dereplication are indicated. (*) Isolated compounds. (**) compounds confirmed by comparison with reference standards. Peaks not highlighted have not been unambiguously identified. Data acquired at electrospray ionization in positive mode (ESI+), UHPLC-DAD-QTOFMS instrument.
    Figure Legend Snippet: BPC chromatogram (100–1000 Da) from the micro-extract of T. wortmannii growth in PDA medium. Compounds identified by UV/Vis-HRMS based dereplication are indicated. (*) Isolated compounds. (**) compounds confirmed by comparison with reference standards. Peaks not highlighted have not been unambiguously identified. Data acquired at electrospray ionization in positive mode (ESI+), UHPLC-DAD-QTOFMS instrument.

    Techniques Used: Isolation

    71) Product Images from "Characteristics and Evolution of sill-driven off-axis hydrothermalism in Guaymas Basin – the Ringvent site"

    Article Title: Characteristics and Evolution of sill-driven off-axis hydrothermalism in Guaymas Basin – the Ringvent site

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-50200-5

    Minerals from the surface and subsurface of Ringvent. (A – F ) Selected images of Ringvent minerals, collected at the seafloor by Alvin . ( A ) diatoms embedded in silica matrix; ( B ) diatom frustule showing partial dissolution; ( C ) opal-A microspheres forming the matrix; ( D ) barite rosette within the matrix formed by opal-A microspheres; ( E,F ) fibrous aragonite cement crystals. Images A to D come from a large silicate sample collected at the ORP site; specimens shown in E and F were collected from a biologically active small mound at the Mound 1 location (Supplemental Fig. 6 ). A and E are photomicrographs of thin sections; B-D and F are SEM microphotographs; analyses were performed by C. Canet and F. Núñez-Useche, UNAM. G-I) Selected images of subsurface silicate. ( G ). Silicate nodule collected at 2.35 m sediment depth in core P11. ( H ), SEM/EDX microphotographs of the nodule surface showing bead-like formations coated with silica at intermediate and ( I ) at high magnification, with barite crystal rosettes on the silica matrix. Analyses were performed by I. Aiello, MLML.
    Figure Legend Snippet: Minerals from the surface and subsurface of Ringvent. (A – F ) Selected images of Ringvent minerals, collected at the seafloor by Alvin . ( A ) diatoms embedded in silica matrix; ( B ) diatom frustule showing partial dissolution; ( C ) opal-A microspheres forming the matrix; ( D ) barite rosette within the matrix formed by opal-A microspheres; ( E,F ) fibrous aragonite cement crystals. Images A to D come from a large silicate sample collected at the ORP site; specimens shown in E and F were collected from a biologically active small mound at the Mound 1 location (Supplemental Fig. 6 ). A and E are photomicrographs of thin sections; B-D and F are SEM microphotographs; analyses were performed by C. Canet and F. Núñez-Useche, UNAM. G-I) Selected images of subsurface silicate. ( G ). Silicate nodule collected at 2.35 m sediment depth in core P11. ( H ), SEM/EDX microphotographs of the nodule surface showing bead-like formations coated with silica at intermediate and ( I ) at high magnification, with barite crystal rosettes on the silica matrix. Analyses were performed by I. Aiello, MLML.

    Techniques Used:

    Geochemical profiles of piston-cored sediments. The plots are contrasting the geochemical profiles of Ringvent core P11 against those from nearby control core P10 and Sonora Margin cores P5/6 and P12. ( A ) δ 13 C-isotopic composition for carbonate nodules and sedimentary carbonate. Porewater δ 13 C-isotopic profiles for ( B ) methane and ( C ) DIC. The range of biogenic methane is based on Guaymas Basin sediment data 31 , 32 ; the range for thermogenic methane is based on δ 13 C-isotopic values near −42‰ for surficial hydrothermal sediments 19 , −43.2 to −50.8‰ in hydrothermal fluid 33 , and −40 to −44‰ in hydrothermal DSDP core 477 30 . ( D ) Porewater sulfate concentrations determined by ion chromatography. ( E ) δ 34 S profiles for porewater sulfate. Complete isotopic data for solid-phase carbonates are tabulated in Supplementary Data 3 . Porewater DIC and methane data are tabulated in Supplementary Data 5A,B , and sulfate and sulfide data (concentrations only for sulfide) are tabulated in Supplementary Data 5C,D .
    Figure Legend Snippet: Geochemical profiles of piston-cored sediments. The plots are contrasting the geochemical profiles of Ringvent core P11 against those from nearby control core P10 and Sonora Margin cores P5/6 and P12. ( A ) δ 13 C-isotopic composition for carbonate nodules and sedimentary carbonate. Porewater δ 13 C-isotopic profiles for ( B ) methane and ( C ) DIC. The range of biogenic methane is based on Guaymas Basin sediment data 31 , 32 ; the range for thermogenic methane is based on δ 13 C-isotopic values near −42‰ for surficial hydrothermal sediments 19 , −43.2 to −50.8‰ in hydrothermal fluid 33 , and −40 to −44‰ in hydrothermal DSDP core 477 30 . ( D ) Porewater sulfate concentrations determined by ion chromatography. ( E ) δ 34 S profiles for porewater sulfate. Complete isotopic data for solid-phase carbonates are tabulated in Supplementary Data 3 . Porewater DIC and methane data are tabulated in Supplementary Data 5A,B , and sulfate and sulfide data (concentrations only for sulfide) are tabulated in Supplementary Data 5C,D .

    Techniques Used: Ion Chromatography

    72) Product Images from "Bi-allelic Mutations in Phe-tRNA Synthetase Associated with a Multi-system Pulmonary Disease Support Non-translational Function"

    Article Title: Bi-allelic Mutations in Phe-tRNA Synthetase Associated with a Multi-system Pulmonary Disease Support Non-translational Function

    Journal: American Journal of Human Genetics

    doi: 10.1016/j.ajhg.2018.06.006

    The c848+1G > A Intronic Mutation Resulted in Aberrant Splicing of FARSB (A) The mutation of the universal splice site motif is expected to affect the splicing of nearby exons in the FARSB gene, such as skipping of the adjacent exon 9. (B) A splice variant that deleted exon 9 of the FARSB _FL transcript, designated as FARSB -ΔE9, was detected in the proband but not in the mother. (C) Quantitative real-time PCR analysis of mRNA expressions of FARSB _E9-11 (using primers targeting exons 9 and 11 of FARSB , thus primarily detecting the full-length transcript), and FARSB -ΔE9 (using primers specifically amplifying the ΔE9 variant) in primary fibroblasts. Gene expression of FARSB _E9-11 and -ΔE9 in the fibroblasts of the proband, both parents, and three control subjects were calculated based on Ct values normalized to house-keeping genes RPL9 and RPS11 . The fold changes were thus calculated by relative to the FARSB _E9-11 level of CTL-17M. Data were presented as mean ± SEM. The ΔE9 RNA was detected only in the proband and father cells but not in the mother and control subjects (U.D. denotes no detectable amplification within 45 qPCR cycles).
    Figure Legend Snippet: The c848+1G > A Intronic Mutation Resulted in Aberrant Splicing of FARSB (A) The mutation of the universal splice site motif is expected to affect the splicing of nearby exons in the FARSB gene, such as skipping of the adjacent exon 9. (B) A splice variant that deleted exon 9 of the FARSB _FL transcript, designated as FARSB -ΔE9, was detected in the proband but not in the mother. (C) Quantitative real-time PCR analysis of mRNA expressions of FARSB _E9-11 (using primers targeting exons 9 and 11 of FARSB , thus primarily detecting the full-length transcript), and FARSB -ΔE9 (using primers specifically amplifying the ΔE9 variant) in primary fibroblasts. Gene expression of FARSB _E9-11 and -ΔE9 in the fibroblasts of the proband, both parents, and three control subjects were calculated based on Ct values normalized to house-keeping genes RPL9 and RPS11 . The fold changes were thus calculated by relative to the FARSB _E9-11 level of CTL-17M. Data were presented as mean ± SEM. The ΔE9 RNA was detected only in the proband and father cells but not in the mother and control subjects (U.D. denotes no detectable amplification within 45 qPCR cycles).

    Techniques Used: Mutagenesis, Variant Assay, Real-time Polymerase Chain Reaction, Expressing, CTL Assay, Amplification

    73) Product Images from "Analysis of Chemical Constituents in Wuzi-Yanzong-Wan by UPLC-ESI-LTQ-Orbitrap-MS"

    Article Title: Analysis of Chemical Constituents in Wuzi-Yanzong-Wan by UPLC-ESI-LTQ-Orbitrap-MS

    Journal: Molecules

    doi: 10.3390/molecules201219765

    Flavonoids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.
    Figure Legend Snippet: Flavonoids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.

    Techniques Used: Mass Spectrometry

    Organic acids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.
    Figure Legend Snippet: Organic acids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.

    Techniques Used: Mass Spectrometry

    Total ion chromatogram (TIC) of WZYZW in negative ion mode ( a ) and positive ion mode ( b ) using UPLC-ESI-LTQ-Orbitrap-MS.
    Figure Legend Snippet: Total ion chromatogram (TIC) of WZYZW in negative ion mode ( a ) and positive ion mode ( b ) using UPLC-ESI-LTQ-Orbitrap-MS.

    Techniques Used: Mass Spectrometry

    Phenylpropanoids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.
    Figure Legend Snippet: Phenylpropanoids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.

    Techniques Used: Mass Spectrometry

    74) Product Images from "Proteins altered by elevated levels of palmitate or glucose implicated in impaired glucose-stimulated insulin secretion"

    Article Title: Proteins altered by elevated levels of palmitate or glucose implicated in impaired glucose-stimulated insulin secretion

    Journal: Proteome Science

    doi: 10.1186/1477-5956-7-24

    INS-1E cell SELDI-TOF mass spectra and 2D-PAGE map . SELDI-TOF mass spectrum (A) and 2D-PAGE map (B) generated from INS-1E cells cultured at 20 mM glucose in the absence or presence of 0.5 mM palmitate for 48 hours. Representative mass spectrum with magnified area of calmodulin with the tentative masses of 16.8, 17.0 and 17.2 kDa, (n = 6) from cells cultured at 20 mM glucose alone (black traces) or presence of palmitate (red traces). Identified proteins are marked in the gel image by their gene names. M r , m/z and pI denote relative molecular mass, mass over charge and isoelectric point of the protein.
    Figure Legend Snippet: INS-1E cell SELDI-TOF mass spectra and 2D-PAGE map . SELDI-TOF mass spectrum (A) and 2D-PAGE map (B) generated from INS-1E cells cultured at 20 mM glucose in the absence or presence of 0.5 mM palmitate for 48 hours. Representative mass spectrum with magnified area of calmodulin with the tentative masses of 16.8, 17.0 and 17.2 kDa, (n = 6) from cells cultured at 20 mM glucose alone (black traces) or presence of palmitate (red traces). Identified proteins are marked in the gel image by their gene names. M r , m/z and pI denote relative molecular mass, mass over charge and isoelectric point of the protein.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Generated, Cell Culture

    75) Product Images from "Disulfide Bond Reduction of Von Willebrand Factor by ADAMTS-13 †"

    Article Title: Disulfide Bond Reduction of Von Willebrand Factor by ADAMTS-13 †

    Journal: Journal of thrombosis and haemostasis : JTH

    doi: 10.1111/j.1538-7836.2010.04094.x

    ADAMTS-13 on newly formed disulfide bonds (A) Sheared VWF (20 nM) was incubated with buffer control; IAA treated or untreated rADAMTS-13 (7 nM) and then mixed with thiol beads (5 mg). VWF was captured by the thiol beads when it was exposed to shear stress in the presence (detected in bead eluate, “e” fraction), but not in the absence of rADAMTS-13 (detected in supernatant, “s” fraction). IAA significantly blocked this rADATMS-13 activity. (B) VWF captured to thiol beads was incubated with buffer control, 15 nM IAA-treated or untreated rADAMTS-13 for 1 hr at 37℃. VWF was released from rADAMTS-13 treated (detected in supernatant, “s” fraction), but not buffer-treated beads (detected in DTT eluate, “e” fraction). Releasing VWF from thiol beads was significantly reduced when IAA treated ADAMTS-13 was used. (C) BSA captured by thiol beads was first treated with buffer control, rADAMTS-13 or IAA-treated rADAMTS-13 (35 nM). After collection of supernatant, BSA bound to thiol beads was eluted with DTT. BSA in both supernatant and DTT eluate was separated on 6% SDS-PAGE and detected by coomassie blue staining (rADAMTS-13 failed to release BSA from the beads (rADAMTS-13 was not visible on the gel image because a > 60 fold dilution of the supernatant [rADAMTS-13
    Figure Legend Snippet: ADAMTS-13 on newly formed disulfide bonds (A) Sheared VWF (20 nM) was incubated with buffer control; IAA treated or untreated rADAMTS-13 (7 nM) and then mixed with thiol beads (5 mg). VWF was captured by the thiol beads when it was exposed to shear stress in the presence (detected in bead eluate, “e” fraction), but not in the absence of rADAMTS-13 (detected in supernatant, “s” fraction). IAA significantly blocked this rADATMS-13 activity. (B) VWF captured to thiol beads was incubated with buffer control, 15 nM IAA-treated or untreated rADAMTS-13 for 1 hr at 37℃. VWF was released from rADAMTS-13 treated (detected in supernatant, “s” fraction), but not buffer-treated beads (detected in DTT eluate, “e” fraction). Releasing VWF from thiol beads was significantly reduced when IAA treated ADAMTS-13 was used. (C) BSA captured by thiol beads was first treated with buffer control, rADAMTS-13 or IAA-treated rADAMTS-13 (35 nM). After collection of supernatant, BSA bound to thiol beads was eluted with DTT. BSA in both supernatant and DTT eluate was separated on 6% SDS-PAGE and detected by coomassie blue staining (rADAMTS-13 failed to release BSA from the beads (rADAMTS-13 was not visible on the gel image because a > 60 fold dilution of the supernatant [rADAMTS-13

    Techniques Used: Incubation, Activity Assay, SDS Page, Staining

    Surface-exposed thiols in ADAMTS-13 (A) rADAMTS-13 (35 nM) was incubated with thiol beads before and after exposure to 100-dyn/cm 2 shear stress for 3 min at 37°C, separated by 6% SDS-PAGE, and detected by immunoblotting with an anti-Myc antibody. rADAMTS-13 was detected in DTT eluate (thiol form), but not in supernatant (disulfide bond form). (B) rADAMTS-13 was first treated with NEM or IAA for 10 min at RT and subjected to dialysis in 1L of 1XPBS to remove excess NEM or IAA. The treated and untreated rADAMTS-13 was then labeled with MPB, separated by 6% SDS-PAGE, and probed with HRP-streptavidin. (C) Normal human plasma (1 ml) was incubated with 100 mM of MPB or captured by 5 mg of thiol beads for 15–20 min at RT. MPB labeled plasma proteins were precipitated by streptavidin beads, released by boiling in SDS sample buffer, separated on 6% SDS-PAGE, and immunoblotted with an anti-ADAMTS-13 antibody (left lane). ADAMTS-13 captured with thiol beads was released by 20 mM DTT, separated on 6% SDS-PAGE, and blotted with an ADAMTS-13 antibody (middle lane). Unlabeled ADAMTS-13 was used as a control (right lane). (D) Normal human plasma (1 ml) was incubated with MPB before and after it was pretreated with 10 mM of IAA. ADAMTS-13 from labeled plasma was captured by streptavidin-coupled sepharose beads, separated on 6% SDS-PAGE, and immunoblotted with an ADAMTS-13 antibody. Because MPB failed to label IAA-treated plasma, streptavidin beads did not precipitate ADAMTS-13 from plasma (lane 2). Plasma without IAA treatment was used as control. (E) Normal human plasma (1 ml) was incubated with an ADAMTS-13 antibody coupled to protein G sepharose beads before and after it was treated with 10 mM of IAA. The bead-captured plasma ADAMTS-13 was able to release VWF from thiol beads. The figure represents 3–6 separate experiments.
    Figure Legend Snippet: Surface-exposed thiols in ADAMTS-13 (A) rADAMTS-13 (35 nM) was incubated with thiol beads before and after exposure to 100-dyn/cm 2 shear stress for 3 min at 37°C, separated by 6% SDS-PAGE, and detected by immunoblotting with an anti-Myc antibody. rADAMTS-13 was detected in DTT eluate (thiol form), but not in supernatant (disulfide bond form). (B) rADAMTS-13 was first treated with NEM or IAA for 10 min at RT and subjected to dialysis in 1L of 1XPBS to remove excess NEM or IAA. The treated and untreated rADAMTS-13 was then labeled with MPB, separated by 6% SDS-PAGE, and probed with HRP-streptavidin. (C) Normal human plasma (1 ml) was incubated with 100 mM of MPB or captured by 5 mg of thiol beads for 15–20 min at RT. MPB labeled plasma proteins were precipitated by streptavidin beads, released by boiling in SDS sample buffer, separated on 6% SDS-PAGE, and immunoblotted with an anti-ADAMTS-13 antibody (left lane). ADAMTS-13 captured with thiol beads was released by 20 mM DTT, separated on 6% SDS-PAGE, and blotted with an ADAMTS-13 antibody (middle lane). Unlabeled ADAMTS-13 was used as a control (right lane). (D) Normal human plasma (1 ml) was incubated with MPB before and after it was pretreated with 10 mM of IAA. ADAMTS-13 from labeled plasma was captured by streptavidin-coupled sepharose beads, separated on 6% SDS-PAGE, and immunoblotted with an ADAMTS-13 antibody. Because MPB failed to label IAA-treated plasma, streptavidin beads did not precipitate ADAMTS-13 from plasma (lane 2). Plasma without IAA treatment was used as control. (E) Normal human plasma (1 ml) was incubated with an ADAMTS-13 antibody coupled to protein G sepharose beads before and after it was treated with 10 mM of IAA. The bead-captured plasma ADAMTS-13 was able to release VWF from thiol beads. The figure represents 3–6 separate experiments.

    Techniques Used: Incubation, SDS Page, Labeling

    Effect of blocking thiols in rADAMTS-13 on VWF cleavage (A) HUVECs were cultured to confluence and then stimulated with 25 mM histamine. Washed platelets with rADAMTS-13 (10 or 20 nM) before and after being treated with 10 mM of IAA (free IAA was removed by dialysis) were perfused over the activated endothelial cells at a 2.5-dyn/cm 2 shear stress for 3 min. The number of ULVWF strings over 10 continuous review fields was then counted and ADAMTS-13 activity was calculated as a percentage of ULVWF strings cleaved during perfusion (Student's t -test, n = 6). (B) Stimulated HUVECs were incubated with lyophilized human platelets in the presence of buffer control, IAA-treated buffer control, rADAMTS-13, or IAA-treated rADAMTS-13. Platelet-decorated ULVWF strings are visible under a light microscope (200 ×, top two panels), but cleaved and undetectable in the presence of IAA-treated and untreated rADAMTS-13 (bottom two panels, bar = 25 μm). (C) Purified plasma VWF multimers were first partially denatured with urea and then incubated with barium-activated rADAMTS-13 with and without IAA treatment. Aliquots were taken at different incubation intervals to detect cleaved VWF fragments by immunoblotting. The figure represents 4 separate experiments.
    Figure Legend Snippet: Effect of blocking thiols in rADAMTS-13 on VWF cleavage (A) HUVECs were cultured to confluence and then stimulated with 25 mM histamine. Washed platelets with rADAMTS-13 (10 or 20 nM) before and after being treated with 10 mM of IAA (free IAA was removed by dialysis) were perfused over the activated endothelial cells at a 2.5-dyn/cm 2 shear stress for 3 min. The number of ULVWF strings over 10 continuous review fields was then counted and ADAMTS-13 activity was calculated as a percentage of ULVWF strings cleaved during perfusion (Student's t -test, n = 6). (B) Stimulated HUVECs were incubated with lyophilized human platelets in the presence of buffer control, IAA-treated buffer control, rADAMTS-13, or IAA-treated rADAMTS-13. Platelet-decorated ULVWF strings are visible under a light microscope (200 ×, top two panels), but cleaved and undetectable in the presence of IAA-treated and untreated rADAMTS-13 (bottom two panels, bar = 25 μm). (C) Purified plasma VWF multimers were first partially denatured with urea and then incubated with barium-activated rADAMTS-13 with and without IAA treatment. Aliquots were taken at different incubation intervals to detect cleaved VWF fragments by immunoblotting. The figure represents 4 separate experiments.

    Techniques Used: Blocking Assay, Cell Culture, Activity Assay, Incubation, Light Microscopy, Purification

    ADAMTS-13 on shear-induced thiol-disulfide exchange of VWF (A) VWF (20 nM) was exposed to a 100-dyn/cm 2 shear stress in the presence and absence of rADAMTS-13 (7 nM) for 3 min at 37°C and incubated with thiol beads for 15–20 min. VWF captured by beads was released by 20 mM DTT and detected by a polyclonal anti-VWF antibody under reducing conditions. (B) VWF multimers were incubated with MPB before and after shear exposure (and with and without rADAMTS-13), and MPB-labeled VWF was detected by HRP-streptavidin. (C) Thiol beads captured VWF before (detected in DTT elute [“e” fraction]), but not after (detected in supernatant [“s” fraction]) being exposed to fluid shear stress in the presence of 1% BSA. (D) VWF was exposed to 100 dyn/cm 2 shear stress in the presence (lane 1) or absence (lane 2) of 7 nM of rADAMTS-13, separated on 6% SDS-PAGE, and immunoblotted with a polyclonal VWF antibody. As a control, VWF was subjected to a standard static assay of VWF cleavage at a low–ionic-strength in the presence of 1 mM BaCl 2 and 1.5 M urea for 0 (lane 3) and 16 hrs (lane 4) at 37°C. The figure represents 3-5 separate experiments.
    Figure Legend Snippet: ADAMTS-13 on shear-induced thiol-disulfide exchange of VWF (A) VWF (20 nM) was exposed to a 100-dyn/cm 2 shear stress in the presence and absence of rADAMTS-13 (7 nM) for 3 min at 37°C and incubated with thiol beads for 15–20 min. VWF captured by beads was released by 20 mM DTT and detected by a polyclonal anti-VWF antibody under reducing conditions. (B) VWF multimers were incubated with MPB before and after shear exposure (and with and without rADAMTS-13), and MPB-labeled VWF was detected by HRP-streptavidin. (C) Thiol beads captured VWF before (detected in DTT elute [“e” fraction]), but not after (detected in supernatant [“s” fraction]) being exposed to fluid shear stress in the presence of 1% BSA. (D) VWF was exposed to 100 dyn/cm 2 shear stress in the presence (lane 1) or absence (lane 2) of 7 nM of rADAMTS-13, separated on 6% SDS-PAGE, and immunoblotted with a polyclonal VWF antibody. As a control, VWF was subjected to a standard static assay of VWF cleavage at a low–ionic-strength in the presence of 1 mM BaCl 2 and 1.5 M urea for 0 (lane 3) and 16 hrs (lane 4) at 37°C. The figure represents 3-5 separate experiments.

    Techniques Used: Incubation, Labeling, SDS Page

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    Article Snippet: .. After incubation overnight at 20°C, ligated RNA was purified (Absolutely RNA kit, Stratagene) and reverse‐transcribed (Superscript III Reverse Transcriptase, Invitrogen). .. PCR amplification of the cDNA library was performed using Phusion polymerase (NEB).

    Article Title: Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1
    Article Snippet: After 8 h incubation, cells were harvested and lysed, followed by the addition of precleared Protein G agarose beads for 1 h. The supernatants were mixed with agarose beads conjugated to anti-HA tag-antibody or control rabbit IgG for 4 h at 4 °C. .. To detect bound MajSAT RNA, 1 μg of precipitated RNA and 5% input were reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen), and semi-quantitative RT-PCR was performed using Mighty Amp DNA polymerase (TaKaRa).

    Article Title: Homeostatic regulation of zinc transporters in the human small intestine by dietary zinc supplementation
    Article Snippet: DNAse I was inactivated by addition of EDTA to 2.5 mM, followed by a 10 minute incubation at 65°C. .. Reverse transcription of poly-A+ RNA (1 μg) or DNAse treated RNA (1 μg) was carried out using Superscript III reverse transcriptase (Invitrogen).

    Expressing:

    Article Title: Imaging cellulose synthase motility during primary cell wall synthesis in the grass Brachypodium distachyon
    Article Snippet: Construction of mEGFP-BdCESA transgenic lines Total RNA was extracted from B. distachyon tissue (7-day-old whole seedlings) and reverse transcribed into cDNA with Invitrogen SuperScript III Reverse Transcriptase. .. A multi-site Gateway reaction united the mEGFP and the CESA coding sequences in the plant expression vector pIPKb002 , in which expression of the tagged CESA genes is driven by a ubiquitin promoter from Zea mays .

    Article Title: Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex *
    Article Snippet: The 9 domains (D1–9) and subdomains (D5a, -b, and -c) of RPM-1 were amplified by RT-PCR from C. elegans total RNA using Superscript III reverse transcriptase (Invitrogen). cDNAs were inserted into pCR8 Topo GY and sequenced to ensure they were mutation-free. .. For transgenic expression of RIP (D5c) in C. elegans , pBG-GY134 (Prgef-1 FLAG GY) was recombined with pBG-GY349 (pCR8 Topo GY RIP (D5c)) to generate pBG-GY440 (Prgef-1 FLAG::RIP (D5c)). pCZ161 encoding full-length RPM-1::GFP driven by its native promoter was engineered to contain a point mutation D2214A (pBG-190).

    Article Title: Brain-Derived Neurotrophic Factor Induces Matrix Metalloproteinase 9 Expression in Neurons via the Serum Response Factor/c-Fos Pathway
    Article Snippet: RNA was reverse transcribed with the SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. .. The following TaqMan Gene Expression Assays (Applied Biosystems) were used: Rn00579162_m1 for MMP-9 and Rn01775763_g1 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; endogenous control).

    Touchdown PCR:

    Article Title: Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci
    Article Snippet: Briefly, 2 μg total RNA from HepG2 cells were reverse transcribed in 20 μl reaction using Superscript III Reverse Transcriptase (Invitrogen, catalogue number 18080044). .. Touchdown-PCR was performed using 2 μl of the cDNA, mixed with 38.75 μl water, 1 μl of each primer (10 μM) (Additional file : Table S8), 1.25 μl dNTP (10 mM), 5 μl 10× Pfu Ultra reaction buffer and 1 μl Pfu Polymerase (Stratagene, catalogue number 600380).

    Western Blot:

    Article Title: Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae
    Article Snippet: Paragraph title: RT–PCR and western blot ... Total RNA was isolated from each sample as described previously , and 5 μg of RNA was reverse transcribed using Superscript III RT enzyme (Invitrogen), as per manufacturer's instructions.

    Transfection:

    Article Title: Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1
    Article Snippet: Briefly, 6 × 106 K512-HAYBX1 cells were seeded on a 10 cm dish, and 8 μg of BrU-labelled MajSAT-Fw RNA was transfected into the cells using TransMessenger reagent (Qiagen). .. To detect bound MajSAT RNA, 1 μg of precipitated RNA and 5% input were reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen), and semi-quantitative RT-PCR was performed using Mighty Amp DNA polymerase (TaKaRa).

    Immunoprecipitation:

    Article Title: Selective termination of lnc RNA transcription promotes heterochromatin silencing and cell differentiation
    Article Snippet: Immunoprecipitated RNA was fragmented 200–300nt long using RNA Fragmentation Reagent Kit from (Ambion). .. After incubation overnight at 20°C, ligated RNA was purified (Absolutely RNA kit, Stratagene) and reverse‐transcribed (Superscript III Reverse Transcriptase, Invitrogen).

    Article Title: Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1
    Article Snippet: Paragraph title: RNA immunoprecipitation ... To detect bound MajSAT RNA, 1 μg of precipitated RNA and 5% input were reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen), and semi-quantitative RT-PCR was performed using Mighty Amp DNA polymerase (TaKaRa).

    Infection:

    Article Title: The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity
    Article Snippet: RNA was extracted from infected cells using the ZR viral RNA kit (Zymo Research) according to the manufacturer's instructions. .. Briefly, 20 μl of extracted RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo), and second-strand synthesis was performed using Sequenase v2.0 (Agilent). cDNA was purified using DNA Clean and Concentrator-5 (Zymo) and subjected to Nextera XT tagmentation (Illumina) followed by 19 cycles of PCR amplification and a 0.8× Ampure XP cleanup (Beckman Coulter).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Transcriptome analysis reveals a comprehensive insect resistance response mechanism in cotton to infestation by the phloem feeding insect Bemisia tabaci (whitefly)
    Article Snippet: Paragraph title: RT‐PCR and qRT‐PCR ... First‐strand cDNA was synthesized from 3 μg of total RNA using SuperScript III Reverse Transcriptase (Invitrogen) in accordance with the manufacturer's instructions and reverse transcribed into cDNA followed by 50× dilution.

    Article Title: Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1
    Article Snippet: .. To detect bound MajSAT RNA, 1 μg of precipitated RNA and 5% input were reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen), and semi-quantitative RT-PCR was performed using Mighty Amp DNA polymerase (TaKaRa). ..

    Article Title: Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae
    Article Snippet: Paragraph title: RT–PCR and western blot ... Total RNA was isolated from each sample as described previously , and 5 μg of RNA was reverse transcribed using Superscript III RT enzyme (Invitrogen), as per manufacturer's instructions.

    Article Title: Homeostatic regulation of zinc transporters in the human small intestine by dietary zinc supplementation
    Article Snippet: Paragraph title: Reverse transcription-polymerase chain reaction (RT-PCR) ... Reverse transcription of poly-A+ RNA (1 μg) or DNAse treated RNA (1 μg) was carried out using Superscript III reverse transcriptase (Invitrogen).

    Article Title: Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex *
    Article Snippet: .. The 9 domains (D1–9) and subdomains (D5a, -b, and -c) of RPM-1 were amplified by RT-PCR from C. elegans total RNA using Superscript III reverse transcriptase (Invitrogen). cDNAs were inserted into pCR8 Topo GY and sequenced to ensure they were mutation-free. .. Clones in pCR8 Topo GY were recombined using LR recombinase with pBG-GY14 to create pBG-GY189 (GFP-D1), pBG-GY190 (GFP-D2), pBG-GY191 (GFP-D3), pBG-GY192 (GFP-D4), pBG-GY193 (GFP-D5), pBG-GY194 (GFP-D6), pBG-GY195 (GFP-D7), pBG-GY196 (GFP-D8), pBG-GY197 (GFP-D9), pBG-GY389 (GFP-D5a), pBG-GY412 (GFP-D5b), and pBG-GY384 (GFP-D5c).

    Generated:

    Article Title: Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1
    Article Snippet: The generated peak list files were used to query either the MSDB database or NCBI using the MASCOT programme ( http://www.matrixscience.com ). .. To detect bound MajSAT RNA, 1 μg of precipitated RNA and 5% input were reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen), and semi-quantitative RT-PCR was performed using Mighty Amp DNA polymerase (TaKaRa).

    Sequencing:

    Article Title: Selective termination of lnc RNA transcription promotes heterochromatin silencing and cell differentiation
    Article Snippet: RNA‐IP experiments coupled to high‐throughput sequencing were conducted in duplicates. .. After incubation overnight at 20°C, ligated RNA was purified (Absolutely RNA kit, Stratagene) and reverse‐transcribed (Superscript III Reverse Transcriptase, Invitrogen).

    Article Title: The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity
    Article Snippet: Paragraph title: Deep sequencing of viral genomes. ... Briefly, 20 μl of extracted RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo), and second-strand synthesis was performed using Sequenase v2.0 (Agilent). cDNA was purified using DNA Clean and Concentrator-5 (Zymo) and subjected to Nextera XT tagmentation (Illumina) followed by 19 cycles of PCR amplification and a 0.8× Ampure XP cleanup (Beckman Coulter).

    Article Title: Brain-Derived Neurotrophic Factor Induces Matrix Metalloproteinase 9 Expression in Neurons via the Serum Response Factor/c-Fos Pathway
    Article Snippet: RNA was reverse transcribed with the SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. .. The cDNA was amplified using a set of custom sequence-specific primers and TaqMan minor groove binder (MGB) probes.

    Binding Assay:

    Article Title: Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1
    Article Snippet: To examine the endogenous binding of YBX1 protein to MajSAT RNA, the RIP assay microRNA kit (MBL) was used according to the manufacturer's protocol. .. To detect bound MajSAT RNA, 1 μg of precipitated RNA and 5% input were reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen), and semi-quantitative RT-PCR was performed using Mighty Amp DNA polymerase (TaKaRa).

    Mutagenesis:

    Article Title: Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex *
    Article Snippet: .. The 9 domains (D1–9) and subdomains (D5a, -b, and -c) of RPM-1 were amplified by RT-PCR from C. elegans total RNA using Superscript III reverse transcriptase (Invitrogen). cDNAs were inserted into pCR8 Topo GY and sequenced to ensure they were mutation-free. .. Clones in pCR8 Topo GY were recombined using LR recombinase with pBG-GY14 to create pBG-GY189 (GFP-D1), pBG-GY190 (GFP-D2), pBG-GY191 (GFP-D3), pBG-GY192 (GFP-D4), pBG-GY193 (GFP-D5), pBG-GY194 (GFP-D6), pBG-GY195 (GFP-D7), pBG-GY196 (GFP-D8), pBG-GY197 (GFP-D9), pBG-GY389 (GFP-D5a), pBG-GY412 (GFP-D5b), and pBG-GY384 (GFP-D5c).

    Isolation:

    Article Title: Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1
    Article Snippet: The pellets were washed four times, and bound RNA was isolated by ethanol precipitation. .. To detect bound MajSAT RNA, 1 μg of precipitated RNA and 5% input were reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen), and semi-quantitative RT-PCR was performed using Mighty Amp DNA polymerase (TaKaRa).

    Article Title: Microarray expression profile analysis of circular RNAs in pancreatic cancer
    Article Snippet: Total RNA from 10 pairs human pancreatic cancer tissue and corresponding paracancerous tissue was isolated using TRIzol reagent (Invitrogen). .. The cDNA synthesis of RNA was performed using SuperScript™ III Reverse Transcriptase (Invitrogen).

    Article Title: Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae
    Article Snippet: .. Total RNA was isolated from each sample as described previously , and 5 μg of RNA was reverse transcribed using Superscript III RT enzyme (Invitrogen), as per manufacturer's instructions. .. The resultant cDNA was PCR amplified for 30 cycle using primers specific for the HML α1, RPB2 , GAL10 gene and the NER genes listed in Supplementary Table 1 .

    Article Title: Brain-Derived Neurotrophic Factor Induces Matrix Metalloproteinase 9 Expression in Neurons via the Serum Response Factor/c-Fos Pathway
    Article Snippet: Total RNA was isolated from 1 × 106 cells using RNeasy Mini kit (Qiagen) as described by the manufacturer. .. RNA was reverse transcribed with the SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions.

    Size-exclusion Chromatography:

    Article Title: Microarray expression profile analysis of circular RNAs in pancreatic cancer
    Article Snippet: The cDNA synthesis of RNA was performed using SuperScript™ III Reverse Transcriptase (Invitrogen). .. The reaction condition was as follows: 95°C for 10 min, 40 cycles of 95°C for 10 sec, 60°C for 60 sec, 95°C for 15 sec.

    Purification:

    Article Title: Selective termination of lnc RNA transcription promotes heterochromatin silencing and cell differentiation
    Article Snippet: .. After incubation overnight at 20°C, ligated RNA was purified (Absolutely RNA kit, Stratagene) and reverse‐transcribed (Superscript III Reverse Transcriptase, Invitrogen). .. PCR amplification of the cDNA library was performed using Phusion polymerase (NEB).

    Article Title: The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity
    Article Snippet: .. Briefly, 20 μl of extracted RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo), and second-strand synthesis was performed using Sequenase v2.0 (Agilent). cDNA was purified using DNA Clean and Concentrator-5 (Zymo) and subjected to Nextera XT tagmentation (Illumina) followed by 19 cycles of PCR amplification and a 0.8× Ampure XP cleanup (Beckman Coulter). .. Libraries were sequenced on a 2 × 300-bp run on an Illumina MiSeq.

    Polymerase Chain Reaction:

    Article Title: Selective termination of lnc RNA transcription promotes heterochromatin silencing and cell differentiation
    Article Snippet: ChIP and RNA‐IP coupled to PCR analysis were performed as described previously (Hiriart et al , ). .. After incubation overnight at 20°C, ligated RNA was purified (Absolutely RNA kit, Stratagene) and reverse‐transcribed (Superscript III Reverse Transcriptase, Invitrogen).

    Article Title: Microarray expression profile analysis of circular RNAs in pancreatic cancer
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation assay for circRNAs ... The cDNA synthesis of RNA was performed using SuperScript™ III Reverse Transcriptase (Invitrogen).

    Article Title: Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae
    Article Snippet: Total RNA was isolated from each sample as described previously , and 5 μg of RNA was reverse transcribed using Superscript III RT enzyme (Invitrogen), as per manufacturer's instructions. .. The resultant cDNA was PCR amplified for 30 cycle using primers specific for the HML α1, RPB2 , GAL10 gene and the NER genes listed in Supplementary Table 1 .

    Article Title: Homeostatic regulation of zinc transporters in the human small intestine by dietary zinc supplementation
    Article Snippet: Reverse transcription of poly-A+ RNA (1 μg) or DNAse treated RNA (1 μg) was carried out using Superscript III reverse transcriptase (Invitrogen). .. Samples were then amplified by PCR using Thermo-Start DNA polymerases (Abgene Ltd, Epsom, Surrey, UK).

    Article Title: The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity
    Article Snippet: .. Briefly, 20 μl of extracted RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo), and second-strand synthesis was performed using Sequenase v2.0 (Agilent). cDNA was purified using DNA Clean and Concentrator-5 (Zymo) and subjected to Nextera XT tagmentation (Illumina) followed by 19 cycles of PCR amplification and a 0.8× Ampure XP cleanup (Beckman Coulter). .. Libraries were sequenced on a 2 × 300-bp run on an Illumina MiSeq.

    Article Title: Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex *
    Article Snippet: The 9 domains (D1–9) and subdomains (D5a, -b, and -c) of RPM-1 were amplified by RT-PCR from C. elegans total RNA using Superscript III reverse transcriptase (Invitrogen). cDNAs were inserted into pCR8 Topo GY and sequenced to ensure they were mutation-free. .. For point mutagenesis, an HpaI-SpeI fragment of RPM-1 was amplified by PCR using pCZ161 as a template and inserted into pCR2.1.

    Article Title: Brain-Derived Neurotrophic Factor Induces Matrix Metalloproteinase 9 Expression in Neurons via the Serum Response Factor/c-Fos Pathway
    Article Snippet: RNA was reverse transcribed with the SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. .. Quantitative real-time PCRs were performed using TaqMan Fast Advanced PCR Master Mix (Applied Biosystems) in an Applied Biosystems 7900HT fast real-time PCR system using the following cycling conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Additionally exemplary products were also visualized on agarose gel.

    Construct:

    Article Title: The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity
    Article Snippet: Metagenomic next-generation sequencing libraries were constructed as described previously ( ). .. Briefly, 20 μl of extracted RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo), and second-strand synthesis was performed using Sequenase v2.0 (Agilent). cDNA was purified using DNA Clean and Concentrator-5 (Zymo) and subjected to Nextera XT tagmentation (Illumina) followed by 19 cycles of PCR amplification and a 0.8× Ampure XP cleanup (Beckman Coulter).

    cDNA Library Assay:

    Article Title: Selective termination of lnc RNA transcription promotes heterochromatin silencing and cell differentiation
    Article Snippet: After incubation overnight at 20°C, ligated RNA was purified (Absolutely RNA kit, Stratagene) and reverse‐transcribed (Superscript III Reverse Transcriptase, Invitrogen). .. PCR amplification of the cDNA library was performed using Phusion polymerase (NEB).

    Chromatin Immunoprecipitation:

    Article Title: Selective termination of lnc RNA transcription promotes heterochromatin silencing and cell differentiation
    Article Snippet: Paragraph title: RNA‐IP and chromatin‐IP ... After incubation overnight at 20°C, ligated RNA was purified (Absolutely RNA kit, Stratagene) and reverse‐transcribed (Superscript III Reverse Transcriptase, Invitrogen).

    Plasmid Preparation:

    Article Title: Imaging cellulose synthase motility during primary cell wall synthesis in the grass Brachypodium distachyon
    Article Snippet: Construction of mEGFP-BdCESA transgenic lines Total RNA was extracted from B. distachyon tissue (7-day-old whole seedlings) and reverse transcribed into cDNA with Invitrogen SuperScript III Reverse Transcriptase. .. A multi-site Gateway reaction united the mEGFP and the CESA coding sequences in the plant expression vector pIPKb002 , in which expression of the tagged CESA genes is driven by a ubiquitin promoter from Zea mays .

    RNA Extraction:

    Article Title: Ultrasensitive Response of Developing Myxococcus xanthus to the Addition of Nutrient Medium Correlates with the Level of MrpC
    Article Snippet: Paragraph title: RNA extraction and analysis. ... Total RNA (1 μg) was used to synthesize cDNA with Superscript III reverse transcriptase (Lifetech) and random primers (Promega), according to the manufacturer's instructions.

    Agarose Gel Electrophoresis:

    Article Title: Brain-Derived Neurotrophic Factor Induces Matrix Metalloproteinase 9 Expression in Neurons via the Serum Response Factor/c-Fos Pathway
    Article Snippet: RNA was reverse transcribed with the SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. .. Quantitative real-time PCRs were performed using TaqMan Fast Advanced PCR Master Mix (Applied Biosystems) in an Applied Biosystems 7900HT fast real-time PCR system using the following cycling conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Additionally exemplary products were also visualized on agarose gel.

    Transgenic Assay:

    Article Title: Imaging cellulose synthase motility during primary cell wall synthesis in the grass Brachypodium distachyon
    Article Snippet: .. Construction of mEGFP-BdCESA transgenic lines Total RNA was extracted from B. distachyon tissue (7-day-old whole seedlings) and reverse transcribed into cDNA with Invitrogen SuperScript III Reverse Transcriptase. ..

    Article Title: Identification of a Peptide Inhibitor of the RPM-1·FSN-1 Ubiquitin Ligase Complex *
    Article Snippet: The 9 domains (D1–9) and subdomains (D5a, -b, and -c) of RPM-1 were amplified by RT-PCR from C. elegans total RNA using Superscript III reverse transcriptase (Invitrogen). cDNAs were inserted into pCR8 Topo GY and sequenced to ensure they were mutation-free. .. For transgenic expression of RIP (D5c) in C. elegans , pBG-GY134 (Prgef-1 FLAG GY) was recombined with pBG-GY349 (pCR8 Topo GY RIP (D5c)) to generate pBG-GY440 (Prgef-1 FLAG::RIP (D5c)). pCZ161 encoding full-length RPM-1::GFP driven by its native promoter was engineered to contain a point mutation D2214A (pBG-190).

    Ethanol Precipitation:

    Article Title: Satellite RNAs promote pancreatic oncogenic processes via the dysfunction of YBX1
    Article Snippet: The pellets were washed four times, and bound RNA was isolated by ethanol precipitation. .. To detect bound MajSAT RNA, 1 μg of precipitated RNA and 5% input were reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen), and semi-quantitative RT-PCR was performed using Mighty Amp DNA polymerase (TaKaRa).

    Next-Generation Sequencing:

    Article Title: The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity
    Article Snippet: Metagenomic next-generation sequencing libraries were constructed as described previously ( ). .. Briefly, 20 μl of extracted RNA was reverse transcribed using SuperScript III reverse transcriptase (Thermo), and second-strand synthesis was performed using Sequenase v2.0 (Agilent). cDNA was purified using DNA Clean and Concentrator-5 (Zymo) and subjected to Nextera XT tagmentation (Illumina) followed by 19 cycles of PCR amplification and a 0.8× Ampure XP cleanup (Beckman Coulter).

    Concentration Assay:

    Article Title: Brain-Derived Neurotrophic Factor Induces Matrix Metalloproteinase 9 Expression in Neurons via the Serum Response Factor/c-Fos Pathway
    Article Snippet: RNA concentration was calculated from the absorbance at 260 nm, and the purity of the RNA was determined by the 260/280-nm absorbance ratio. .. RNA was reverse transcribed with the SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions.

    High Throughput Screening Assay:

    Article Title: Selective termination of lnc RNA transcription promotes heterochromatin silencing and cell differentiation
    Article Snippet: RNA‐IP experiments coupled to high‐throughput sequencing were conducted in duplicates. .. After incubation overnight at 20°C, ligated RNA was purified (Absolutely RNA kit, Stratagene) and reverse‐transcribed (Superscript III Reverse Transcriptase, Invitrogen).

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    Thermo Fisher mouse anti gfap
    <t>IL-1β</t> signal is colocalized with <t>GFAP,</t> and not with Iba-1 or NeuN, in the dorsal hippocampus in stressed and non-stressed animals A. Representative images of IL-1β, NeuN, Iba-1, and GFAP immunoreactivity in the dentate gyrus of the DH (AP −3.36 mm from bregma) acquired at 20X are shown. Because we did not detect any differences in colocalization between stressed and non-stressed rats, all images here are taken from animals that received stress exposure. Bitplane Imaris was used for background subtraction to better visualize individual cells presented. B. Bitplane Imaris software was used to calculate the colocalization of the IL-1β signal with GFAP, Iba-1, and NeuN. Colocalization analyses revealed that the percent of the IL-1β signal colocalized with GFAP was significantly greater than the percent of the IL-1β signal colocalized with either Iba-1 or NeuN. * p
    Mouse Anti Gfap, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cyclosporine a
    Effect of <t>cyclosporine-A</t> or GaNP on biofilm forming potential of M.tuberculosis H 37 Rv cells: H 37 Rv cells expressing PpiB were grown in static culture in absence or presence of cyclosporine-A (100 µg/ml) or GaNP (25, 50 µg/ml) for 7 days, as described in materials and methods. Representative figure of the pellicle formed at the liquid-air interface is shown. Suppressive effect of cyclosporine-A a and GaNP b on biofilm formation in M.tuberculosis H 37 Rv strains is evident. c Pretreatment or posttreatment of H 37 Rv cells with GaNP and the resultant suppression in biofilm formation
    Cyclosporine A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher acarbose
    Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, <t>acarbose</t> and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively
    Acarbose, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher sfpq
    <t>BCL9</t> regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d <t>SFPQ</t> binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P
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    Image Search Results


    IL-1β signal is colocalized with GFAP, and not with Iba-1 or NeuN, in the dorsal hippocampus in stressed and non-stressed animals A. Representative images of IL-1β, NeuN, Iba-1, and GFAP immunoreactivity in the dentate gyrus of the DH (AP −3.36 mm from bregma) acquired at 20X are shown. Because we did not detect any differences in colocalization between stressed and non-stressed rats, all images here are taken from animals that received stress exposure. Bitplane Imaris was used for background subtraction to better visualize individual cells presented. B. Bitplane Imaris software was used to calculate the colocalization of the IL-1β signal with GFAP, Iba-1, and NeuN. Colocalization analyses revealed that the percent of the IL-1β signal colocalized with GFAP was significantly greater than the percent of the IL-1β signal colocalized with either Iba-1 or NeuN. * p

    Journal: Brain, behavior, and immunity

    Article Title: Hippocampal interleukin-1 mediates stress-enhanced fear learning: A potential role for astrocyte-derived interleukin-1β

    doi: 10.1016/j.bbi.2017.09.016

    Figure Lengend Snippet: IL-1β signal is colocalized with GFAP, and not with Iba-1 or NeuN, in the dorsal hippocampus in stressed and non-stressed animals A. Representative images of IL-1β, NeuN, Iba-1, and GFAP immunoreactivity in the dentate gyrus of the DH (AP −3.36 mm from bregma) acquired at 20X are shown. Because we did not detect any differences in colocalization between stressed and non-stressed rats, all images here are taken from animals that received stress exposure. Bitplane Imaris was used for background subtraction to better visualize individual cells presented. B. Bitplane Imaris software was used to calculate the colocalization of the IL-1β signal with GFAP, Iba-1, and NeuN. Colocalization analyses revealed that the percent of the IL-1β signal colocalized with GFAP was significantly greater than the percent of the IL-1β signal colocalized with either Iba-1 or NeuN. * p

    Article Snippet: The following primary antibodies were used: rabbit anti-IL-1β (1:500, Abcam, Cambridge, MA, Cat# Ab9722), mouse anti-GFAP (1:1000, ThermoFisher Scientific, Waltham, MA, Cat #MS-1376P), mouse anti-NeuN-Alexa 568 (1:1000, Abcam Cambridge, MA, ), and rabbit anti-Iba-1-biotinylated (1:500, Wako, Richmond, VA, Cat#016-26461).

    Techniques: Software

    Effect of cyclosporine-A or GaNP on biofilm forming potential of M.tuberculosis H 37 Rv cells: H 37 Rv cells expressing PpiB were grown in static culture in absence or presence of cyclosporine-A (100 µg/ml) or GaNP (25, 50 µg/ml) for 7 days, as described in materials and methods. Representative figure of the pellicle formed at the liquid-air interface is shown. Suppressive effect of cyclosporine-A a and GaNP b on biofilm formation in M.tuberculosis H 37 Rv strains is evident. c Pretreatment or posttreatment of H 37 Rv cells with GaNP and the resultant suppression in biofilm formation

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Effect of cyclosporine-A or GaNP on biofilm forming potential of M.tuberculosis H 37 Rv cells: H 37 Rv cells expressing PpiB were grown in static culture in absence or presence of cyclosporine-A (100 µg/ml) or GaNP (25, 50 µg/ml) for 7 days, as described in materials and methods. Representative figure of the pellicle formed at the liquid-air interface is shown. Suppressive effect of cyclosporine-A a and GaNP b on biofilm formation in M.tuberculosis H 37 Rv strains is evident. c Pretreatment or posttreatment of H 37 Rv cells with GaNP and the resultant suppression in biofilm formation

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Expressing

    Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Concentration Assay, Activity Assay

    Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb PpiB (Rv2582) exhibits homology with proteins from other biofilm forming bacteria and possesses similar amino acids Arg and Pro at the binding site of cyclosporine-A (highlighted in green box) and acarbose (highlighted in red box), respectively. A dimer of atomic gallium 17 similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb PpiB (Rv2582) exhibits homology with proteins from other biofilm forming bacteria and possesses similar amino acids Arg and Pro at the binding site of cyclosporine-A (highlighted in green box) and acarbose (highlighted in red box), respectively. A dimer of atomic gallium 17 similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Sequencing, Binding Assay, SPR Assay

    Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Mass Spectrometry, Incubation, Cell Culture, Alamar Blue Assay

    Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Crystal Violet Assay, Mass Spectrometry, Cell Culture, Incubation

    Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Schematic overview of the effect of repurposed drugs on biofilm and its outcome on tuberculosis treatment: Under stress like conditions Mycobacteria secrete exogenous layer of matrix that forms a physical barrier for entry of drugs. The cells within the matrix continuously secrete to develop a biomass of biofilm that enables the cells to withstand high minimum inhibitory concentration (MIC) of drugs. As a result, higher dosage of drugs is required to kill the cells. Cells at the core of the biofilm matrix are least affected by drugs and evolve in due time so as to withstand even higher concentration of drugs. This confers drug tolerance and leads to drug toxicity, increased treatment cost and mortality. Cyclosporine-A, acarbose and GaNP inhibit the activity of PpiB that play crucial role in biofilm formation. Treatment with these drugs suppresses formation of biofilm and the bacterium is exposed directly to the drugs. As a result the drug is effective at low MIC values. Treatment with these drugs also reduces the MIC of existing anti-tubercular drugs resulting in decreased toxicity. The end result is that patient mortality and treatment cost may be reduced significantly. Regular and dotted arrows in the figure denote confirmed and putative roles respectively

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Concentration Assay, Activity Assay

    Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Multiple sequence alignment of M.tb PpiB in biofilm forming bacteria and interaction of PpiB with cyclosporine-A, acarbose and GaNP. a M.tb similarly binds to Gly residue (highlighted in black box), which is conserved within the PpiB binding site of all biofilm-forming bacteria. b , d , f Interaction of cyclosporine-A, acarbose and dimer of atomic gallium with PpiB was tested by molecular docking analysis. b The docked complex of cyclosporine-A and PpiB. The protein (pink) is shown in surface view whereas interacting residues (grey) and ligand (green) is represented in stick model. Hydrogen bond (yellow) is shown in dotted lines. d Interactions of PpiB with acarbose showing various hydrogen and hydrophobic interactions. f The docked complex of dimer of atomic gallium and PpiB. The protein (pink) is shown in surface view whereas interacting residues (green) and ligand (red) is represented in stick model. Hydrogen bond (black) is shown in dotted lines. c , e , g SPR analysis was performed as described in methods. Response units (RU) of the interaction of PpiB with cyclosporine-A ( c ) or acrabose e , or GaNP g from representative experiment are shown

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Sequencing, Binding Assay, SPR Assay

    Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Effect of anti-TB drugs on the survival of M. smegmatis in the presence and absence of cyclosporine-A or acarbose or GaNP. Ms_VC and Ms_PpiB cells were induced with anhydrotetracycline to express ppiase in absence and presence of cyclosporine-A (100 μg/ml) a , d or acarbose (1000 μg/ml) b or GaNP (50 μg/ml) c , e . Cells were incubated in static culture to allow biofilm formation. At the end of 7 days Ms _ VC and Ms_PpiB, cultured in absence of anhydrotetracycline [( )VC tet-, ( ) PpiB tet-] or presence of anhydrotetracycline [( ) VC tet+, ( ) PpiB tet+], were treated either with isoniazid (0, 8, 16, 32, 64 μg/ml) or ethambutol (0, 0.25, 1, 4, 16 μg/ml) and further incubated for 68 h. Susceptibility of M. smegmatis to isoniazid in absence and presence of biofilm was scored by assessing the viability of cells in a 4 h alamar blue assay. Values shown from a representative experiment are means [±s.e.m] of percent cell viability

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Mass Spectrometry, Incubation, Cell Culture, Alamar Blue Assay

    Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p

    Journal: NPJ Biofilms and Microbiomes

    Article Title: Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention

    doi: 10.1038/s41522-018-0075-0

    Figure Lengend Snippet: Effect of cyclosporine-A, acarbose or GaNP on induction of biofilm in M. smegmatis (Crystal violet assay). Ms_VC and Ms_PpiB cells were cultured in absence [( )VC tet-, ( ) PpiB tet-] or presence [( ) VC tet+, ( ) PpiB tet+] of anhydrotetracycline, as described in methods. Cells were treated with cyclosporine-A (0, 10, 100, 1000 μg/ml) a or acarbose (0, 1, 10, 100, 500, 1000 μg/ml) b or GaNP (0, 10, 50, 100, 1000 μg/ml) c and incubated for 7 days. At the end point, biofilm was quantified as described in methods. Values shown from a representative experiment are means [±s.e.m] of biofilm formed.* p

    Article Snippet: Anhydrotetracycline induced Ms_PpiB and Ms_VC cells were cultured in the presence of various concentrations of cyclosporine-A (0, 10, 100, and 1000 µg/ml), acarbose (0, 1, 10, 100, 500, 1000 µg/ml) or GaNP (0, 10, 50, 100, 1000 µg/ml) in sterile flat bottom 96-well microtiter plate (Thermo Scientific, India).

    Techniques: Crystal Violet Assay, Mass Spectrometry, Cell Culture, Incubation

    BCL9 regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d SFPQ binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P

    Journal: Nature Communications

    Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins

    doi: 10.1038/s41467-019-13842-7

    Figure Lengend Snippet: BCL9 regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d SFPQ binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P

    Article Snippet: The membrane was subsequently probed using anti-BCL9 (1:1000 dilution, H00000607-M01, Abnova), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), or β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories) antibodies.

    Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Functional Assay, Binding Assay, Knock-Out