cytosolic fractions  (Thermo Fisher)


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    Structured Review

    Thermo Fisher cytosolic fractions
    Heat maps of differentially expressed proteins in miR-378-spg CHO cells. A and B show the clustering of significantly increased and decreased proteins identified in the <t>cytosolic</t> enriched fraction of miR-378-spg cells for day 4 and day 8, respectively. C and D show the clustering of differentially expressed proteins identified in the membrane enriched fraction of miR-378-spg when compared to control on day 4 and day 8 of culture, respectively. The normalised abundance values of differentially expressed proteins were log2 transformed and hierarchical Pearson clustering was performed on Z-score normalised intensity values.
    Cytosolic Fractions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3689 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A proteomic profiling dataset of recombinant Chinese hamster ovary cells showing enhanced cellular growth following miR-378 depletion"

    Article Title: A proteomic profiling dataset of recombinant Chinese hamster ovary cells showing enhanced cellular growth following miR-378 depletion

    Journal: Data in Brief

    doi: 10.1016/j.dib.2018.11.115

    Heat maps of differentially expressed proteins in miR-378-spg CHO cells. A and B show the clustering of significantly increased and decreased proteins identified in the cytosolic enriched fraction of miR-378-spg cells for day 4 and day 8, respectively. C and D show the clustering of differentially expressed proteins identified in the membrane enriched fraction of miR-378-spg when compared to control on day 4 and day 8 of culture, respectively. The normalised abundance values of differentially expressed proteins were log2 transformed and hierarchical Pearson clustering was performed on Z-score normalised intensity values.
    Figure Legend Snippet: Heat maps of differentially expressed proteins in miR-378-spg CHO cells. A and B show the clustering of significantly increased and decreased proteins identified in the cytosolic enriched fraction of miR-378-spg cells for day 4 and day 8, respectively. C and D show the clustering of differentially expressed proteins identified in the membrane enriched fraction of miR-378-spg when compared to control on day 4 and day 8 of culture, respectively. The normalised abundance values of differentially expressed proteins were log2 transformed and hierarchical Pearson clustering was performed on Z-score normalised intensity values.

    Techniques Used: Transformation Assay

    2) Product Images from "Protective Effect of a (Poly)phenol-Rich Extract Derived from Sweet Cherries Culls against Oxidative Cell Damage"

    Article Title: Protective Effect of a (Poly)phenol-Rich Extract Derived from Sweet Cherries Culls against Oxidative Cell Damage

    Journal: Molecules

    doi: 10.3390/molecules21040406

    Chromatographic profiles of Ch-PRE ( A1 ) High Performance Liquid Chromatography coupled with a diode-array detector (HPLC-DAD) profile at 280 nm; ( A2 ) HPLC-DAD profile at 527 nm; and ( B ) HPLC coupled with a DAD and electrochemical detector (ED) (HPLC-DAD-ED). Legend: 1—Neochlorogenic acid, 2—Catechin, 3—Chlorogenic acid, 4—Procyanidin B2, 5—Cyanidin-3-glucoside, 6—Cyanidin-3-rutinoside, 7—Peonidin-3-glucoside, 8—Quercetin-3-rutinoside, 9—Quercetin-3-glucoside, 10—Kaempferol-3-glucoside, 11—Sakuranin, 12—Isosakuranetin.
    Figure Legend Snippet: Chromatographic profiles of Ch-PRE ( A1 ) High Performance Liquid Chromatography coupled with a diode-array detector (HPLC-DAD) profile at 280 nm; ( A2 ) HPLC-DAD profile at 527 nm; and ( B ) HPLC coupled with a DAD and electrochemical detector (ED) (HPLC-DAD-ED). Legend: 1—Neochlorogenic acid, 2—Catechin, 3—Chlorogenic acid, 4—Procyanidin B2, 5—Cyanidin-3-glucoside, 6—Cyanidin-3-rutinoside, 7—Peonidin-3-glucoside, 8—Quercetin-3-rutinoside, 9—Quercetin-3-glucoside, 10—Kaempferol-3-glucoside, 11—Sakuranin, 12—Isosakuranetin.

    Techniques Used: High Performance Liquid Chromatography

    3) Product Images from "PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65"

    Article Title: PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

    Journal: Open Biology

    doi: 10.1098/rsob.120080

    Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.
    Figure Legend Snippet: Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.

    Techniques Used: Mass Spectrometry, Activation Assay, Expressing, Transfection, Lysis, Immunoprecipitation, SDS Page, Staining, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

    4) Product Images from "Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1"

    Article Title: Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1

    Journal: Molecular Systems Biology

    doi: 10.1002/msb.201304750

    A systematic affinity purification mass spectrometry ( AP ‐ MS ) approach to define the human Hpo pathway interaction proteome Selection of primary and secondary baits in this study. Baits were selected sequentially, starting with the core components of the Hpo kinase signaling pathway and extended based on obtained AP ‐ MS results or homology to D rosophila H po components. Biochemical workflow for native protein complex purification from HEK 293‐Flp T‐rex cells. Bait proteins were expressed from a tetracycline‐inducible CMV promoter, with a N‐terminal Strep‐ HA fusion tag following induction with doxycycline for 24 h. Cells were lysed, complexes affinity‐purified and processed for analysis by tandem mass spectrometry. Data analysis pipeline. Acquired mass spectra from 90 experiments (at least 2 biological replicates per bait) were searched with X!Tandem. Search results were statistically validated by the Trans‐Proteomic Pipeline ( TPP ) to match a protein identification false discovery rate of
    Figure Legend Snippet: A systematic affinity purification mass spectrometry ( AP ‐ MS ) approach to define the human Hpo pathway interaction proteome Selection of primary and secondary baits in this study. Baits were selected sequentially, starting with the core components of the Hpo kinase signaling pathway and extended based on obtained AP ‐ MS results or homology to D rosophila H po components. Biochemical workflow for native protein complex purification from HEK 293‐Flp T‐rex cells. Bait proteins were expressed from a tetracycline‐inducible CMV promoter, with a N‐terminal Strep‐ HA fusion tag following induction with doxycycline for 24 h. Cells were lysed, complexes affinity‐purified and processed for analysis by tandem mass spectrometry. Data analysis pipeline. Acquired mass spectra from 90 experiments (at least 2 biological replicates per bait) were searched with X!Tandem. Search results were statistically validated by the Trans‐Proteomic Pipeline ( TPP ) to match a protein identification false discovery rate of

    Techniques Used: Affinity Purification, Mass Spectrometry, Selection, Purification

    5) Product Images from "Use of Physiologically Based Biokinetic (PBBK) Modeling to Study Estragole Bioactivation and Detoxification in Humans as Compared with Male Rats"

    Article Title: Use of Physiologically Based Biokinetic (PBBK) Modeling to Study Estragole Bioactivation and Detoxification in Humans as Compared with Male Rats

    Journal: Toxicological Sciences

    doi: 10.1093/toxsci/kfp102

    (A) Chromatographic profile of an incubation with human liver microsomes at a substrate concentration of 500μM 1′-hydroxyestragole, NAD + as cofactor and GSH to trap the transient 1′-oxoestragole at an incubation time of 10 min. (B) 1′-Hydroxyestragole concentration dependent rate of formation of 1′-oxoestragole by human (filled triangle) and male rat (filled circle) liver microsomes as determined by quantification of the GS-1′-oxoestragole, which is assumed to represent the formation of 1′-oxoestragole. In the plots each point represents the mean (± SD) of three replicates.
    Figure Legend Snippet: (A) Chromatographic profile of an incubation with human liver microsomes at a substrate concentration of 500μM 1′-hydroxyestragole, NAD + as cofactor and GSH to trap the transient 1′-oxoestragole at an incubation time of 10 min. (B) 1′-Hydroxyestragole concentration dependent rate of formation of 1′-oxoestragole by human (filled triangle) and male rat (filled circle) liver microsomes as determined by quantification of the GS-1′-oxoestragole, which is assumed to represent the formation of 1′-oxoestragole. In the plots each point represents the mean (± SD) of three replicates.

    Techniques Used: Incubation, Concentration Assay

    6) Product Images from "Resveratrol, but not EGCG, in the diet suppresses DMBA-induced mammary cancer in rats"

    Article Title: Resveratrol, but not EGCG, in the diet suppresses DMBA-induced mammary cancer in rats

    Journal: Journal of Carcinogenesis

    doi: 10.1186/1477-3163-5-15

    Tumor latency (2A, B, C) in female Sprague-Dawley CD rats exposed to resveratrol, EGCG, or AIN-76A (control) from birth until sacrifice . On day 50 postpartum, all animals were treated with 60 mg DMBA/kg body weight. Figures 2A, 2B, and 2C depict the time to first tumor, second tumor, and third tumor (latency), respectively. A p value
    Figure Legend Snippet: Tumor latency (2A, B, C) in female Sprague-Dawley CD rats exposed to resveratrol, EGCG, or AIN-76A (control) from birth until sacrifice . On day 50 postpartum, all animals were treated with 60 mg DMBA/kg body weight. Figures 2A, 2B, and 2C depict the time to first tumor, second tumor, and third tumor (latency), respectively. A p value

    Techniques Used:

    Tumor multiplicity in female Sprague-Dawley CD rats exposed to resveratrol, EGCG, or AIN-76A (control) from birth until sacrifice . On day 50 postpartum, all animals were treated with 60 mg DMBA/kg body weight. Figure 1 shows tumor multiplicity, with the values representing mean tumors per rat ± 2 standard errors. A p value
    Figure Legend Snippet: Tumor multiplicity in female Sprague-Dawley CD rats exposed to resveratrol, EGCG, or AIN-76A (control) from birth until sacrifice . On day 50 postpartum, all animals were treated with 60 mg DMBA/kg body weight. Figure 1 shows tumor multiplicity, with the values representing mean tumors per rat ± 2 standard errors. A p value

    Techniques Used:

    7) Product Images from "Serratia marcescens secretes proteases and chitinases with larvicidal activity against Anopheles dirus"

    Article Title: Serratia marcescens secretes proteases and chitinases with larvicidal activity against Anopheles dirus

    Journal: bioRxiv

    doi: 10.1101/2020.05.31.123539

    Larvicidal molecule(s) secreted by S. marcescens are heat sensitive macromolecule(s). (A) Heat sensitivity of larvicidal activity from S. marcescens spent medium. The S. marcescens spent medium was heated at 45, 70, and 95 °C for one hour before testing for larvicidal activity at 10% (v/v) final concentration. LB medium was used as a control at a final concentration of 10% (v/v). The experiment was performed in duplicate (B) Larvicidal activity of S. marcescens spent medium after separation of small (
    Figure Legend Snippet: Larvicidal molecule(s) secreted by S. marcescens are heat sensitive macromolecule(s). (A) Heat sensitivity of larvicidal activity from S. marcescens spent medium. The S. marcescens spent medium was heated at 45, 70, and 95 °C for one hour before testing for larvicidal activity at 10% (v/v) final concentration. LB medium was used as a control at a final concentration of 10% (v/v). The experiment was performed in duplicate (B) Larvicidal activity of S. marcescens spent medium after separation of small (

    Techniques Used: Activity Assay, Concentration Assay

    Protease inhibitor reduced larvicidal activity of S. marcescens secreted macromolecule. The experiment was performed in four replicates. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R.
    Figure Legend Snippet: Protease inhibitor reduced larvicidal activity of S. marcescens secreted macromolecule. The experiment was performed in four replicates. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R.

    Techniques Used: Protease Inhibitor, Activity Assay

    S. marcescens secreted larvicidal molecules when cultured under static condition at 25 °C but not 37 °C. (A) Survival curve of An. dirus larvae after treatment with S. marcescens spent media cultured under static condition at 25 °C or 37 °C. The experiment was performed in triplicate. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R. (B) SDS-PAGE of S. marcescens spent media cultured under static condition at 25 °C or 37 °C. Two red arrows indicate major protein bands that were expressed when the bacterium was cultured at 25 °C but not 37 °C.
    Figure Legend Snippet: S. marcescens secreted larvicidal molecules when cultured under static condition at 25 °C but not 37 °C. (A) Survival curve of An. dirus larvae after treatment with S. marcescens spent media cultured under static condition at 25 °C or 37 °C. The experiment was performed in triplicate. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R. (B) SDS-PAGE of S. marcescens spent media cultured under static condition at 25 °C or 37 °C. Two red arrows indicate major protein bands that were expressed when the bacterium was cultured at 25 °C but not 37 °C.

    Techniques Used: Cell Culture, SDS Page

    C6/36 cell viability assay of E. coli spent medium (Ech_c), S. marcescens spent medium (Ser_m), and 5 μM cycloheximide. The cell viability was shown relative to LB medium treatment. Spent medium and LB were used at 10% (v/v) final concentration. P-values were calculated using raw data from sulforhodamine B assay by One-way ANOVA with TukeyHSD post hoc test with multcompView package in R software.
    Figure Legend Snippet: C6/36 cell viability assay of E. coli spent medium (Ech_c), S. marcescens spent medium (Ser_m), and 5 μM cycloheximide. The cell viability was shown relative to LB medium treatment. Spent medium and LB were used at 10% (v/v) final concentration. P-values were calculated using raw data from sulforhodamine B assay by One-way ANOVA with TukeyHSD post hoc test with multcompView package in R software.

    Techniques Used: Viability Assay, Concentration Assay, Sulforhodamine B Assay, Software

    Intrinsic larvicidal activity of S. marcescens macromolecule and its enhancing effect on larvae’s susceptibility to bacterial infection. (A) Survival of An. dirus larvae treated with 2X concentration of macromolecules from S. marcescens spent medium diluted in LB with or without antibiotics. LB with or without antibiotics were used as a control. The experiment was performed in duplicate. (B): An. dirus larvae treated with S. marcescens macromolecules reconstituted to the final concentration of 0.5X, 1X, and 2X in PBS. The experiment was performed in duplicate. (C): Treatment of sublethal dose of S. marcescens secreted macromolecule enhances An. dirus larvae susceptibility to bacterial infection. The larvae were treated for 24 hours with 0.5x concentration of macromolecules from S. marcescens diluted in PBS buffer before addition of Pseudomonas sp. at final concentration of OD 600 = 0.08, 0.2, 0.4, 1, and 5. Mortality of the larvae was monitored every 3 hours for 24 hours. The experiment was performed in triplicates. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R.
    Figure Legend Snippet: Intrinsic larvicidal activity of S. marcescens macromolecule and its enhancing effect on larvae’s susceptibility to bacterial infection. (A) Survival of An. dirus larvae treated with 2X concentration of macromolecules from S. marcescens spent medium diluted in LB with or without antibiotics. LB with or without antibiotics were used as a control. The experiment was performed in duplicate. (B): An. dirus larvae treated with S. marcescens macromolecules reconstituted to the final concentration of 0.5X, 1X, and 2X in PBS. The experiment was performed in duplicate. (C): Treatment of sublethal dose of S. marcescens secreted macromolecule enhances An. dirus larvae susceptibility to bacterial infection. The larvae were treated for 24 hours with 0.5x concentration of macromolecules from S. marcescens diluted in PBS buffer before addition of Pseudomonas sp. at final concentration of OD 600 = 0.08, 0.2, 0.4, 1, and 5. Mortality of the larvae was monitored every 3 hours for 24 hours. The experiment was performed in triplicates. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R.

    Techniques Used: Activity Assay, Infection, Concentration Assay

    Survival analyses of An. dirus mosquito larvae after treatment with bacterial spent media at 10% (v/v) concentration. (A) Survival curve of An. dirus larvae after treatment with bacterial spent media cultured under planktonic condition or static condition. The spent media were collected at three days after incubation under planktonic or static state at 25 °C Ech_c: Escherichia coli strain XL10, Aci_s: Acinetobactor soli , Leu_spp: Leucobactor spp., Och_spp: Ochrobatrum spp., Ser_m: Serratia marcescens , Sph_m: Sphingobacterium multivorum , Sph_spp: Sphingobacterium spp., Ste_m: Stenotrophomonas maltophilia . LB medium or water was used as a control at final concentration of 10% (v/v). The experiment was performed in triplicate (B) Survival curve of An. dirus larvae after treatment with S. marcescens spent medium cultured under planktonic or static condition and collected after 1, 2, or 3 days after inoculation. The experiment was performed in duplicate (C) Survival curve of An. dirus larvae after treatment with E. coli or S. marcescens cells at various concentration. Bacterial cells were cultured under static condition at 25 °C then washed twice with PBS. The experiment was performed in triplicate. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R. Significance level: **: p
    Figure Legend Snippet: Survival analyses of An. dirus mosquito larvae after treatment with bacterial spent media at 10% (v/v) concentration. (A) Survival curve of An. dirus larvae after treatment with bacterial spent media cultured under planktonic condition or static condition. The spent media were collected at three days after incubation under planktonic or static state at 25 °C Ech_c: Escherichia coli strain XL10, Aci_s: Acinetobactor soli , Leu_spp: Leucobactor spp., Och_spp: Ochrobatrum spp., Ser_m: Serratia marcescens , Sph_m: Sphingobacterium multivorum , Sph_spp: Sphingobacterium spp., Ste_m: Stenotrophomonas maltophilia . LB medium or water was used as a control at final concentration of 10% (v/v). The experiment was performed in triplicate (B) Survival curve of An. dirus larvae after treatment with S. marcescens spent medium cultured under planktonic or static condition and collected after 1, 2, or 3 days after inoculation. The experiment was performed in duplicate (C) Survival curve of An. dirus larvae after treatment with E. coli or S. marcescens cells at various concentration. Bacterial cells were cultured under static condition at 25 °C then washed twice with PBS. The experiment was performed in triplicate. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R. Significance level: **: p

    Techniques Used: Concentration Assay, Cell Culture, Incubation

    S. marcescens secreted macromolecules have chitinase activity and chitinase inhibitor reduced larvicidal activity. (A) S. marcescens secreted chitinase that resulted in a clear zone around bacterium colony when cultured at 25 °C but not 37 °C. (B) S. marcescens spent medium accumulated chitinase activity over time and saturated after three days of incubation. One unit of chitinase activity was defined as the amount of enzyme which releases 1 μg of reducing sugar per hour as measured by pHBAH assay. (C) Cyclo-Gly-Pro inhibit chitinase activity in a dose-dependent manner. Statistical analyses comparing chitinase activity and inhibition of chitinase activity were performed using One-way ANOVA with Tukey multiple comparison test using multcompView package in R. (D) Chitinase inhibitor reduced larvicidal activity of the S. marcescens secreted macromolecule. The experiment was performed in duplicate. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R.
    Figure Legend Snippet: S. marcescens secreted macromolecules have chitinase activity and chitinase inhibitor reduced larvicidal activity. (A) S. marcescens secreted chitinase that resulted in a clear zone around bacterium colony when cultured at 25 °C but not 37 °C. (B) S. marcescens spent medium accumulated chitinase activity over time and saturated after three days of incubation. One unit of chitinase activity was defined as the amount of enzyme which releases 1 μg of reducing sugar per hour as measured by pHBAH assay. (C) Cyclo-Gly-Pro inhibit chitinase activity in a dose-dependent manner. Statistical analyses comparing chitinase activity and inhibition of chitinase activity were performed using One-way ANOVA with Tukey multiple comparison test using multcompView package in R. (D) Chitinase inhibitor reduced larvicidal activity of the S. marcescens secreted macromolecule. The experiment was performed in duplicate. Statistical significance of the survival analyses was calculated using Log-rank test followed by a Bonferroni post-hoc analysis using ‘survminer ‘package in R.

    Techniques Used: Activity Assay, Cell Culture, Incubation, Inhibition

    8) Product Images from "Global Screening of Sentrin-Specific Protease Family Substrates in SUMOylation"

    Article Title: Global Screening of Sentrin-Specific Protease Family Substrates in SUMOylation

    Journal: bioRxiv

    doi: 10.1101/2020.02.25.964072

    Strategy of SUMO1 modified sites immunoaffinity purification. (A) Representation of the C-terminal sequence comparison of SUMO1 WT , SUMO1 ΔGG and SUMO1 T95R after trypsin digestion. (B) Immunoblot analysis confirmed the expressions of SUMO1-conjugated proteins in SUMO1 WT and SUMO1 T95R . HEK293T cells were stably transfected with indicated plasmids and SUMO1 ΔGG was chosen as the negative control. Ponceau-S staining was shown as loading control. (C) Schematic overview of His 10 -SUMO1 T95R -modified peptides identification. Stably expressing cell samples transfected with indicated plasmids were subjected to the first concentration of SUMOylated proteins and trypsin digestion, followed by peptide IP to enrich SUMO1-conjugated peptides. diGly featured peptides were subsequently exposed for LC-MS/MS identification. (D) Validation of expression patterns and level of conjugation by immunoblot analysis. His 10 -SUMO1 WT , His 10 -SUMO1 ΔGG , SUMO1 T95R and SUMO1 T95K stable cells were lysed and subjected to Ni-NTA pulldown assay. The presence of ubiquitin and ubiquitin-like modifier patterns were confirmed with indicated antibodies. (E) Detection of SUMOylation patterns after transfection of SENP family members. SUMOylated proteins from His 10 -SUMO1 T95R stably expressed cells with EV and Flag-tagged SENP family members transfected were pulled down and analyzed by immunoblot using anti-SUMO1, anti-Flag or anti-Actin antibodies. Ponceau-S staining was shown as loading control. (F) Schematic workflow of the deduction strategy for constructing SUMO1-modified proteome. Venn plot indicated the number of overlapped diGly sites detected in His 10 -SUMO1 ΔGG , SUMO1 T95R and SUMO1 T95K stable cells.
    Figure Legend Snippet: Strategy of SUMO1 modified sites immunoaffinity purification. (A) Representation of the C-terminal sequence comparison of SUMO1 WT , SUMO1 ΔGG and SUMO1 T95R after trypsin digestion. (B) Immunoblot analysis confirmed the expressions of SUMO1-conjugated proteins in SUMO1 WT and SUMO1 T95R . HEK293T cells were stably transfected with indicated plasmids and SUMO1 ΔGG was chosen as the negative control. Ponceau-S staining was shown as loading control. (C) Schematic overview of His 10 -SUMO1 T95R -modified peptides identification. Stably expressing cell samples transfected with indicated plasmids were subjected to the first concentration of SUMOylated proteins and trypsin digestion, followed by peptide IP to enrich SUMO1-conjugated peptides. diGly featured peptides were subsequently exposed for LC-MS/MS identification. (D) Validation of expression patterns and level of conjugation by immunoblot analysis. His 10 -SUMO1 WT , His 10 -SUMO1 ΔGG , SUMO1 T95R and SUMO1 T95K stable cells were lysed and subjected to Ni-NTA pulldown assay. The presence of ubiquitin and ubiquitin-like modifier patterns were confirmed with indicated antibodies. (E) Detection of SUMOylation patterns after transfection of SENP family members. SUMOylated proteins from His 10 -SUMO1 T95R stably expressed cells with EV and Flag-tagged SENP family members transfected were pulled down and analyzed by immunoblot using anti-SUMO1, anti-Flag or anti-Actin antibodies. Ponceau-S staining was shown as loading control. (F) Schematic workflow of the deduction strategy for constructing SUMO1-modified proteome. Venn plot indicated the number of overlapped diGly sites detected in His 10 -SUMO1 ΔGG , SUMO1 T95R and SUMO1 T95K stable cells.

    Techniques Used: Modification, Immunoaffinity Purification, Sequencing, Stable Transfection, Transfection, Negative Control, Staining, Expressing, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Conjugation Assay

    SENP3 promotes antiviral immunity during poly (I:C) stimulation in HEK293 cells. (A) Schematic representations of SENP3 interactome identification during poly (I:C) stimulation in HEK293 cells. EV and Flag-tagged SENP3 were transfected into HEK293 cells and poly (I:C) stimulation was performed 6hr before cell collection. The lysates were subjected to immunoprecipitation and then in gel digestion for LC-MS/MS. Three biological replicates were performed for data analysis. (B) The interaction network of SENP3 in HEK293 cells, with or without poly (I:C) treatment. (C) Increased association of SENP3 interacted inflammatory proteins under poly (I:C) stimulation and the expression profiles were represented. (D) The GO/pathway enrichment analysis of SENP3 interactome under poly (I:C) stimulation. (E) Potential mechanism of SENP3 in regulating RNA-virus induced immune signaling pathway.
    Figure Legend Snippet: SENP3 promotes antiviral immunity during poly (I:C) stimulation in HEK293 cells. (A) Schematic representations of SENP3 interactome identification during poly (I:C) stimulation in HEK293 cells. EV and Flag-tagged SENP3 were transfected into HEK293 cells and poly (I:C) stimulation was performed 6hr before cell collection. The lysates were subjected to immunoprecipitation and then in gel digestion for LC-MS/MS. Three biological replicates were performed for data analysis. (B) The interaction network of SENP3 in HEK293 cells, with or without poly (I:C) treatment. (C) Increased association of SENP3 interacted inflammatory proteins under poly (I:C) stimulation and the expression profiles were represented. (D) The GO/pathway enrichment analysis of SENP3 interactome under poly (I:C) stimulation. (E) Potential mechanism of SENP3 in regulating RNA-virus induced immune signaling pathway.

    Techniques Used: Transfection, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Expressing

    9) Product Images from "Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta"

    Article Title: Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta

    Journal: bioRxiv

    doi: 10.1101/802892

    Gene expression in aEC1 cells is largely independent of S1P/S1PR1 signaling. (A) Pie chart of all aEC1-enriched transcripts (versus aEC2-6) indicating those which were also differentially expressed in S1pr1-ECKO MAECs. (B) Heatmap (row Z-scores) of the 20 transcripts that were both S1PR1-regulated and aEC1-enriched (left). Expression of these transcripts in each cluster from scRNA-seq analysis is shown (right). (C) Gene expression in aEC1 was compared to all ECs (LEC, vEC, and aEC2-6 collectively). All transcripts expressed greater than 16-fold higher in aEC1 are shown (25 transcripts total). Red, blue and black transcript names indicate up- regulated, down-regulated or similar levels of expression in S1pr1-ECKO MAECs, respectively. (D) Immunostaining of S1pr1-ECKO and WT aortae whole mount en face preparations for ITGA6 and VEC. Images are representative of observations from two pairs of animals (N=4). (E) ITGA6 and VEC immunostaining of whole mount en face preparations of thoracic aortae from S1PR1-GS mice bearing Sphk2 -/- Sphk1 f/f or Sphk2 -/- Sphk1 f/f Rosa26-Cre-ER T2 alleles. (F) Quantification of GFP+ arterial EC at branch point (n=12) and non-branch point (n=6) locations (n=2 mice for each genotype). Branch point EC were defined as cells included in the first three rows around the edge of orifices. Only GFP+ EC in the same Z-plane as surrounding arterial EC were counted (GFP+ EC of intercostal arteries were in a different Z-plane and therefore were not counted as branch point EC). All scale bars are 100 µM.
    Figure Legend Snippet: Gene expression in aEC1 cells is largely independent of S1P/S1PR1 signaling. (A) Pie chart of all aEC1-enriched transcripts (versus aEC2-6) indicating those which were also differentially expressed in S1pr1-ECKO MAECs. (B) Heatmap (row Z-scores) of the 20 transcripts that were both S1PR1-regulated and aEC1-enriched (left). Expression of these transcripts in each cluster from scRNA-seq analysis is shown (right). (C) Gene expression in aEC1 was compared to all ECs (LEC, vEC, and aEC2-6 collectively). All transcripts expressed greater than 16-fold higher in aEC1 are shown (25 transcripts total). Red, blue and black transcript names indicate up- regulated, down-regulated or similar levels of expression in S1pr1-ECKO MAECs, respectively. (D) Immunostaining of S1pr1-ECKO and WT aortae whole mount en face preparations for ITGA6 and VEC. Images are representative of observations from two pairs of animals (N=4). (E) ITGA6 and VEC immunostaining of whole mount en face preparations of thoracic aortae from S1PR1-GS mice bearing Sphk2 -/- Sphk1 f/f or Sphk2 -/- Sphk1 f/f Rosa26-Cre-ER T2 alleles. (F) Quantification of GFP+ arterial EC at branch point (n=12) and non-branch point (n=6) locations (n=2 mice for each genotype). Branch point EC were defined as cells included in the first three rows around the edge of orifices. Only GFP+ EC in the same Z-plane as surrounding arterial EC were counted (GFP+ EC of intercostal arteries were in a different Z-plane and therefore were not counted as branch point EC). All scale bars are 100 µM.

    Techniques Used: Expressing, Immunostaining, Mouse Assay

    S1P is required for S1PR1/ß-arrestin coupling in LECs of aorta-associated lymphatics. (A) Confocal images of a sagittal cryosection (14 µM) of an S1RP1-GS mouse aorta immunostained for VE-Cadherin and Lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1). White arrows indicate GFP+ arterial ECs at a branch point orifices and yellow arrows indicate adventitia-associated GFP+ LECs. Scale bar is 50 µM. (B-C) Confocal images of whole- mount preparations of S1RP1-GS mouse thoracic aortae immunostained for Vascular endothelial growth factor receptor 3 (VEGFR3; Flt4 ), LYVE1, and CLDN5 with the tunica intima (B) or tunica adventitia (C) in contact with coverslip. Orange stars indicate VEGFR3+LYVE1+GFP+ areas, cyan stars indicate VEGFR3+LYVE1 low GFP+ areas, and magenta stars indicate VEGFR3+LYVE1+GFP- areas. Scale bars are 100 µM (B) and 50 µM (C) . (D) Representative images of mesentery lymphatics from S1PR1-GS mice bearing Sphk2 -/- : Sphk1 f/f (n=3) or Sphk2 -/- : Sphk1 f/f : Lyve1-Cre (n=3) alleles whole mounted and immunostained for PROX1. Solid arrows: GFP+PROX1+, dashed arrows: GFP-PROX1+, arrowheads: GFP+PROX1- (arterioles). Scale bar is 100 µM. (E) Quantification of GFP+ signal over PROX1+ areas (N=6). Scale bars are 100 µM.
    Figure Legend Snippet: S1P is required for S1PR1/ß-arrestin coupling in LECs of aorta-associated lymphatics. (A) Confocal images of a sagittal cryosection (14 µM) of an S1RP1-GS mouse aorta immunostained for VE-Cadherin and Lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1). White arrows indicate GFP+ arterial ECs at a branch point orifices and yellow arrows indicate adventitia-associated GFP+ LECs. Scale bar is 50 µM. (B-C) Confocal images of whole- mount preparations of S1RP1-GS mouse thoracic aortae immunostained for Vascular endothelial growth factor receptor 3 (VEGFR3; Flt4 ), LYVE1, and CLDN5 with the tunica intima (B) or tunica adventitia (C) in contact with coverslip. Orange stars indicate VEGFR3+LYVE1+GFP+ areas, cyan stars indicate VEGFR3+LYVE1 low GFP+ areas, and magenta stars indicate VEGFR3+LYVE1+GFP- areas. Scale bars are 100 µM (B) and 50 µM (C) . (D) Representative images of mesentery lymphatics from S1PR1-GS mice bearing Sphk2 -/- : Sphk1 f/f (n=3) or Sphk2 -/- : Sphk1 f/f : Lyve1-Cre (n=3) alleles whole mounted and immunostained for PROX1. Solid arrows: GFP+PROX1+, dashed arrows: GFP-PROX1+, arrowheads: GFP+PROX1- (arterioles). Scale bar is 100 µM. (E) Quantification of GFP+ signal over PROX1+ areas (N=6). Scale bars are 100 µM.

    Techniques Used: Mouse Assay

    10) Product Images from "Differential proteomic analysis of synovial fluid from rheumatoid arthritis and osteoarthritis patients"

    Article Title: Differential proteomic analysis of synovial fluid from rheumatoid arthritis and osteoarthritis patients

    Journal: Clinical proteomics

    doi: 10.1186/1559-0275-11-1

    A schematic workflow illustrating the steps involved in the differential analysis of RA and OA synovial fluid proteome. Proteins from RA and OA synovial fluid were extracted and depleted to remove the 14 most abundant proteins using multiple affinity removal system, Human-14. The depleted protein from RA and OA were then digested with trypsin and labeled with iTRAQ reagents, 117 and 116 respectively. The labeled samples were pooled and subjected to fractionation using strong cation exchange chromatography. The fractions were then analyzed on a LTQ-Orbitrap Velos mass spectrometer. The MS/MS data obtained was searched against Human RefSeq 50 database using Sequest and Mascot search algorithms. Validation of the iTRAQ quantitation data was carried out using multiple reaction monitoring and Western blot.
    Figure Legend Snippet: A schematic workflow illustrating the steps involved in the differential analysis of RA and OA synovial fluid proteome. Proteins from RA and OA synovial fluid were extracted and depleted to remove the 14 most abundant proteins using multiple affinity removal system, Human-14. The depleted protein from RA and OA were then digested with trypsin and labeled with iTRAQ reagents, 117 and 116 respectively. The labeled samples were pooled and subjected to fractionation using strong cation exchange chromatography. The fractions were then analyzed on a LTQ-Orbitrap Velos mass spectrometer. The MS/MS data obtained was searched against Human RefSeq 50 database using Sequest and Mascot search algorithms. Validation of the iTRAQ quantitation data was carried out using multiple reaction monitoring and Western blot.

    Techniques Used: Labeling, Fractionation, Chromatography, Mass Spectrometry, Quantitation Assay, Western Blot

    11) Product Images from "Escherichia coli Quorum-Sensing EDF, A Peptide Generated by Novel Multiple Distinct Mechanisms and Regulated by trans-Translation"

    Article Title: Escherichia coli Quorum-Sensing EDF, A Peptide Generated by Novel Multiple Distinct Mechanisms and Regulated by trans-Translation

    Journal: mBio

    doi: 10.1128/mBio.02034-15

    Truncation by MazF of zwf mRNA at WT positions −2 and 701 leads to the generation of EDF. (A) The nucleotide and amino acid sequences of the section of zwf that is involved in the generation of EDF. The sequence of the pentapeptide EDF is shown in a bold, red rectangle. Point mutations that prevented the generation of EDF are shown in yellow; point mutations that did not prevent the generation of EDF are shown in green. Upstream from the EDF-encoding region, the codon regions which, when deleted or replaced, prevented EDF generation are shown in magenta rectangles. Immediately upstream from the EDF coding region, the 3 amino acids which, when mutated, prevented the generation of EDF are shown in a blue rectangle. The stretch of codons between the EDF-encoding sequence and the MazF cleavage site, ACA at position 701, is shown in a black rectangle. (B) Viability assay showing the role in EDF generation of MazF ACA cleavage sites at zwf positions −2 and 701. To prepare dense culture supernatants (SNs), we used the E. coli MC4100 relA + (WT) strain (lane a) or its deletion mutation derivative, the MC4100relA + Δ zwf strain, carrying plasmid p zwf (lane b), p zwf −2 AAA (lane c), p zwf 701 AAA (lane d), or p zwf 706 TAA (lane e). Each of these strains was grown in M9 medium without or with ampicillin. When each culture reached a density of 3 × 10 8 cells/ml, we prepared the SN; we determined the activity of each SN by viability assay (see Materials and Methods). (C) High-performance liquid chromatography (HPLC) analysis showing the role in EDF generation of MazF ACA cleavage sites at zwf positions −2 and 701. SNs were prepared as described above for panel B. We determined the presence of EDF in these dense culture SNs by measuring the absorbance at 220 nm by HPLC (see Materials and Methods). The EDF retention time, marked by an arrow, was 9.07 min, which was determined using a synthetic EDF peptide as a standard (see Fig. S1Be in the supplemental material and Materials and Methods). (D) Primer extension analysis showing that MazF cleaves in vitro zwf mRNA at the ACA site located at position 701. zwf was transcribed in vitro , and its MazF cleavage sites were determined by primer extension as described in Materials and Methods. (i) Sequencing (lanes 1 to 4) and primer extension (lane 5) analysis using in vitro -transcribed zwf mRNA employing a primer (D27) which binds to nucleotides 749 to 766 of the zwf mRNA. Lanes 6 and 7 show the results of primer extension analysis performed with primer D27, employing in vitro -transcribed zwf mRNA that was incubated either in the absence or in the presence of purified MazF as specified in Materials and Methods. The signal that indicates the MazF cleavage at position 701 is indicated (black arrow). The colored stars to the left indicate signals which originated from termination of primer extension due to the mRNA structures shown in panel ii. (ii) Secondary structure analysis performed using the program Mfold ( 62 ) and drawn with the program VARNA ( 63 ) of the zwf region shown in panel i. The ACA site at position 701 is shown in red, and the MazF cleavage is indicated (black arrow). The structures that result in premature stop of reverse transcription (shown in panel i) are indicated by the corresponding colored stars.
    Figure Legend Snippet: Truncation by MazF of zwf mRNA at WT positions −2 and 701 leads to the generation of EDF. (A) The nucleotide and amino acid sequences of the section of zwf that is involved in the generation of EDF. The sequence of the pentapeptide EDF is shown in a bold, red rectangle. Point mutations that prevented the generation of EDF are shown in yellow; point mutations that did not prevent the generation of EDF are shown in green. Upstream from the EDF-encoding region, the codon regions which, when deleted or replaced, prevented EDF generation are shown in magenta rectangles. Immediately upstream from the EDF coding region, the 3 amino acids which, when mutated, prevented the generation of EDF are shown in a blue rectangle. The stretch of codons between the EDF-encoding sequence and the MazF cleavage site, ACA at position 701, is shown in a black rectangle. (B) Viability assay showing the role in EDF generation of MazF ACA cleavage sites at zwf positions −2 and 701. To prepare dense culture supernatants (SNs), we used the E. coli MC4100 relA + (WT) strain (lane a) or its deletion mutation derivative, the MC4100relA + Δ zwf strain, carrying plasmid p zwf (lane b), p zwf −2 AAA (lane c), p zwf 701 AAA (lane d), or p zwf 706 TAA (lane e). Each of these strains was grown in M9 medium without or with ampicillin. When each culture reached a density of 3 × 10 8 cells/ml, we prepared the SN; we determined the activity of each SN by viability assay (see Materials and Methods). (C) High-performance liquid chromatography (HPLC) analysis showing the role in EDF generation of MazF ACA cleavage sites at zwf positions −2 and 701. SNs were prepared as described above for panel B. We determined the presence of EDF in these dense culture SNs by measuring the absorbance at 220 nm by HPLC (see Materials and Methods). The EDF retention time, marked by an arrow, was 9.07 min, which was determined using a synthetic EDF peptide as a standard (see Fig. S1Be in the supplemental material and Materials and Methods). (D) Primer extension analysis showing that MazF cleaves in vitro zwf mRNA at the ACA site located at position 701. zwf was transcribed in vitro , and its MazF cleavage sites were determined by primer extension as described in Materials and Methods. (i) Sequencing (lanes 1 to 4) and primer extension (lane 5) analysis using in vitro -transcribed zwf mRNA employing a primer (D27) which binds to nucleotides 749 to 766 of the zwf mRNA. Lanes 6 and 7 show the results of primer extension analysis performed with primer D27, employing in vitro -transcribed zwf mRNA that was incubated either in the absence or in the presence of purified MazF as specified in Materials and Methods. The signal that indicates the MazF cleavage at position 701 is indicated (black arrow). The colored stars to the left indicate signals which originated from termination of primer extension due to the mRNA structures shown in panel ii. (ii) Secondary structure analysis performed using the program Mfold ( 62 ) and drawn with the program VARNA ( 63 ) of the zwf region shown in panel i. The ACA site at position 701 is shown in red, and the MazF cleavage is indicated (black arrow). The structures that result in premature stop of reverse transcription (shown in panel i) are indicated by the corresponding colored stars.

    Techniques Used: Sequencing, Viability Assay, Mutagenesis, Plasmid Preparation, Activity Assay, High Performance Liquid Chromatography, In Vitro, Incubation, Purification

    12) Product Images from "Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis"

    Article Title: Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    Journal: Marine Drugs

    doi: 10.3390/md13074331

    Specific and volumetric scopularide A production by S. brevicaulis LF580 and transformants TF1-5-1 and TF5-2, which contain the gene encoding the putative transcription factor for the NRPS1-PKS2 gene cluster under a constitutive promoter, when grown in ( a ) defined medium and ( b ) complex medium. Error bars represent ± sem ( n = 2 for TF1-5-1 and n = 2 for LF580 in defined medium, n = 3 for LF580 in complex medium [ 16 ]).
    Figure Legend Snippet: Specific and volumetric scopularide A production by S. brevicaulis LF580 and transformants TF1-5-1 and TF5-2, which contain the gene encoding the putative transcription factor for the NRPS1-PKS2 gene cluster under a constitutive promoter, when grown in ( a ) defined medium and ( b ) complex medium. Error bars represent ± sem ( n = 2 for TF1-5-1 and n = 2 for LF580 in defined medium, n = 3 for LF580 in complex medium [ 16 ]).

    Techniques Used:

    Structure of scopularide A and the related compounds emericellamide A and W493-B.
    Figure Legend Snippet: Structure of scopularide A and the related compounds emericellamide A and W493-B.

    Techniques Used:

    ( A ) Chromatogram of a scopularide A of the lowest concentration of standard (0.02 µg/mL) used in the experiments; ( B ) Standard curve for scopularide A.
    Figure Legend Snippet: ( A ) Chromatogram of a scopularide A of the lowest concentration of standard (0.02 µg/mL) used in the experiments; ( B ) Standard curve for scopularide A.

    Techniques Used: Concentration Assay

    Proposed biosynthetic pathway for scopularide A using emericellamide and W493-B as models [ 14 , 15 ].
    Figure Legend Snippet: Proposed biosynthetic pathway for scopularide A using emericellamide and W493-B as models [ 14 , 15 ].

    Techniques Used:

    13) Product Images from "Obesity is associated with changes in oxysterol metabolism and levels in mice liver, hypothalamus, adipose tissue and plasma"

    Article Title: Obesity is associated with changes in oxysterol metabolism and levels in mice liver, hypothalamus, adipose tissue and plasma

    Journal: Scientific Reports

    doi: 10.1038/srep19694

    Plasmatic oxysterol levels during the development of diet-induced obesity. Oxysterol levels were quantified by HPLC-MS in mouse plasma of the diet-induced obesity model. At each time-point (i.e. 1; 2; 4; 6; 8 and 16 weeks) a control and a high-fat group were sacrificed. The data are reported as percentage of each respective control group (shown as a dotted line). Data are mean ± s.e.m. Student’s t-test between HFD group and the respective CTL group. *P
    Figure Legend Snippet: Plasmatic oxysterol levels during the development of diet-induced obesity. Oxysterol levels were quantified by HPLC-MS in mouse plasma of the diet-induced obesity model. At each time-point (i.e. 1; 2; 4; 6; 8 and 16 weeks) a control and a high-fat group were sacrificed. The data are reported as percentage of each respective control group (shown as a dotted line). Data are mean ± s.e.m. Student’s t-test between HFD group and the respective CTL group. *P

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry, CTL Assay

    Hepatic oxysterol levels and expression of their metabolic enzymes during the development of diet-induced obesity. Schematic representation of the main metabolic steps involved in the synthesis and degradation of the oxysterols measured throughout the HFD study in the liver. The figure shows the variations in oxysterol levels and mRNA expression of their metabolic enzymes, compared to the respective control mice, at the different time-points throughout the study. At each time-point (i.e. 1; 2; 4; 6; 8 and 16 weeks) a control and a high-fat group were sacrificed. Oxysterol levels were quantified by HPLC-MS and mRNA enzyme expression was measured by qRT-PCR. Red color indicates an increase and blue color indicates a decrease of the HFD group compared to control group. Data (mean ± s.e.m) are reported in Table S2 . Student’s t-test between HFD group and the respective CTL group. *P
    Figure Legend Snippet: Hepatic oxysterol levels and expression of their metabolic enzymes during the development of diet-induced obesity. Schematic representation of the main metabolic steps involved in the synthesis and degradation of the oxysterols measured throughout the HFD study in the liver. The figure shows the variations in oxysterol levels and mRNA expression of their metabolic enzymes, compared to the respective control mice, at the different time-points throughout the study. At each time-point (i.e. 1; 2; 4; 6; 8 and 16 weeks) a control and a high-fat group were sacrificed. Oxysterol levels were quantified by HPLC-MS and mRNA enzyme expression was measured by qRT-PCR. Red color indicates an increase and blue color indicates a decrease of the HFD group compared to control group. Data (mean ± s.e.m) are reported in Table S2 . Student’s t-test between HFD group and the respective CTL group. *P

    Techniques Used: Expressing, Mouse Assay, High Performance Liquid Chromatography, Mass Spectrometry, Quantitative RT-PCR, CTL Assay

    Oxysterol levels in the liver, plasma and adipose tissue of the ob/ob genetic model of obesity. Oxysterol levels were quantified by HPLC-MS in ( a ) the liver, ( b ) the plasma and ( c ) the adipose tissue in the ob/ob model. The data are reported as percentage of the control group (ob/lean) (shown as a dotted line). Data are mean ± s.e.m.; student’s t-test between ob/ob group and the ob/lean group *P
    Figure Legend Snippet: Oxysterol levels in the liver, plasma and adipose tissue of the ob/ob genetic model of obesity. Oxysterol levels were quantified by HPLC-MS in ( a ) the liver, ( b ) the plasma and ( c ) the adipose tissue in the ob/ob model. The data are reported as percentage of the control group (ob/lean) (shown as a dotted line). Data are mean ± s.e.m.; student’s t-test between ob/ob group and the ob/lean group *P

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry

    Adipose tissue oxysterol levels during the development of diet-induced obesity. Oxysterol levels were quantified by HPLC-MS in mouse subcutaneous adipose tissue of the diet-induced obesity model. At each time-point (i.e. 1; 2; 4; 6; 8 and 16 weeks) a control and a high-fat group were sacrificed. The data are reported as percentage of each respective control group (shown as a dotted line). Data are mean ± s.e.m. Student’s t-test between HFD group and the respective CTL group. *P
    Figure Legend Snippet: Adipose tissue oxysterol levels during the development of diet-induced obesity. Oxysterol levels were quantified by HPLC-MS in mouse subcutaneous adipose tissue of the diet-induced obesity model. At each time-point (i.e. 1; 2; 4; 6; 8 and 16 weeks) a control and a high-fat group were sacrificed. The data are reported as percentage of each respective control group (shown as a dotted line). Data are mean ± s.e.m. Student’s t-test between HFD group and the respective CTL group. *P

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry, CTL Assay

    Hypothalamic oxysterol levels and expression of their metabolic enzymes during the development of diet-induced obesity. Schematic representation of the main metabolic steps involved in the synthesis and degradation of the oxysterols measured throughout the HFD study in the hypothalamus. The figure shows the variations in oxysterol levels and mRNA expression of their metabolic enzymes, compared to the respective control mice, at the different time-points throughout the study. At each time-point (i.e. 1; 2; 4; 6; 8 and 16 weeks) a normal diet (control) and a high-fat group were sacrificed. Oxysterol levels were quantified by HPLC-MS and mRNA enzyme expression was measured by qRT-PCR. Red color indicates an increase and blue color indicates a decrease of the HFD group compared to control group. Data (mean ± s.e.m) are reported in Table S1 . Student’s t-test between HFD group and the respective CTL group. *P
    Figure Legend Snippet: Hypothalamic oxysterol levels and expression of their metabolic enzymes during the development of diet-induced obesity. Schematic representation of the main metabolic steps involved in the synthesis and degradation of the oxysterols measured throughout the HFD study in the hypothalamus. The figure shows the variations in oxysterol levels and mRNA expression of their metabolic enzymes, compared to the respective control mice, at the different time-points throughout the study. At each time-point (i.e. 1; 2; 4; 6; 8 and 16 weeks) a normal diet (control) and a high-fat group were sacrificed. Oxysterol levels were quantified by HPLC-MS and mRNA enzyme expression was measured by qRT-PCR. Red color indicates an increase and blue color indicates a decrease of the HFD group compared to control group. Data (mean ± s.e.m) are reported in Table S1 . Student’s t-test between HFD group and the respective CTL group. *P

    Techniques Used: Expressing, Mouse Assay, High Performance Liquid Chromatography, Mass Spectrometry, Quantitative RT-PCR, CTL Assay

    14) Product Images from "LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography"

    Article Title: LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography

    Journal: Journal of Proteome Research

    doi: 10.1021/pr500570m

    Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant differences in expressions between DF versus EAC with p value
    Figure Legend Snippet: Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant differences in expressions between DF versus EAC with p value

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Spectral counts quantitation of LAC-enriched glycoproteins (A) and HC-enriched glycoproteins (B) by LC–ESI–MS/MS that were evaluated as significant differentiated ones between DF and HGD and between DF and EAC with p value
    Figure Legend Snippet: Spectral counts quantitation of LAC-enriched glycoproteins (A) and HC-enriched glycoproteins (B) by LC–ESI–MS/MS that were evaluated as significant differentiated ones between DF and HGD and between DF and EAC with p value

    Techniques Used: Quantitation Assay, Mass Spectrometry

    Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant difference between DF versus EAC and DF versus HGD with p value
    Figure Legend Snippet: Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant difference between DF versus EAC and DF versus HGD with p value

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Principal component analysis (PCA) scores plot for LC–ESI–MS/MS results of LAC- (A) and HC-enriched (B) samples from human blood serum associated with DF subjects ( N = 15, green), HGD ( N = 12 for LAC enrichment and N = 11 for HC enrichment, blue), and EAC ( N = 15, red).
    Figure Legend Snippet: Principal component analysis (PCA) scores plot for LC–ESI–MS/MS results of LAC- (A) and HC-enriched (B) samples from human blood serum associated with DF subjects ( N = 15, green), HGD ( N = 12 for LAC enrichment and N = 11 for HC enrichment, blue), and EAC ( N = 15, red).

    Techniques Used: Mass Spectrometry

    Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant differences in expressions between DF versus HGD with p value
    Figure Legend Snippet: Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant differences in expressions between DF versus HGD with p value

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    15) Product Images from "Label-Free Proteomics Assisted by Affinity Enrichment for Elucidating the Chemical Reactivity of the Liver Mitochondrial Proteome toward Adduction by the Lipid Electrophile 4-hydroxy-2-nonenal (HNE)"

    Article Title: Label-Free Proteomics Assisted by Affinity Enrichment for Elucidating the Chemical Reactivity of the Liver Mitochondrial Proteome toward Adduction by the Lipid Electrophile 4-hydroxy-2-nonenal (HNE)

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2016.00002

    Heatmap visualization of quantitative values of the 182 proteins enriched at the protein level . Left: heatmap of the 182 proteins identified and quantified over all six HNE exposure groups; Right: Zoomed-in region of the heatmap focusing on the most abundantly detected and identified putative HNE protein adducts. After affinity capture, samples were trypsin-digested and analyzed using LC-MS. Protein quantification was based on the peak intensity of the 3 most intense peptides. A total of 182 proteins were quantified. From top to bottom the 182 identified proteins are listed and each row represents a protein and its corresponding abundance. From left to right, HNE concentrations that were used for the in vitro exposure experiments of the mitochondrial protein samples. The color in each cell represents protein abundance obtained from the “Hi3” peptide intensity approach: red is more abundant and dark green is less abundant. Black indicates missing values.
    Figure Legend Snippet: Heatmap visualization of quantitative values of the 182 proteins enriched at the protein level . Left: heatmap of the 182 proteins identified and quantified over all six HNE exposure groups; Right: Zoomed-in region of the heatmap focusing on the most abundantly detected and identified putative HNE protein adducts. After affinity capture, samples were trypsin-digested and analyzed using LC-MS. Protein quantification was based on the peak intensity of the 3 most intense peptides. A total of 182 proteins were quantified. From top to bottom the 182 identified proteins are listed and each row represents a protein and its corresponding abundance. From left to right, HNE concentrations that were used for the in vitro exposure experiments of the mitochondrial protein samples. The color in each cell represents protein abundance obtained from the “Hi3” peptide intensity approach: red is more abundant and dark green is less abundant. Black indicates missing values.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, In Vitro

    16) Product Images from "Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex"

    Article Title: Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002222

    Quantification of calcium-dependent regulation of phosphorylation sites of Toxoplasma invasion motor complex components. A ) Work flow to identify individual phosphorylation sites and quantitatively assess their responsiveness to calcium signals using a SILAC-based proteomics approach. A 1∶1 mixture of Triton X-100 lysates from “Heavy” (H; Arg4/Lys8)-labeled ethanol-stimulated tachyzoites or
    Figure Legend Snippet: Quantification of calcium-dependent regulation of phosphorylation sites of Toxoplasma invasion motor complex components. A ) Work flow to identify individual phosphorylation sites and quantitatively assess their responsiveness to calcium signals using a SILAC-based proteomics approach. A 1∶1 mixture of Triton X-100 lysates from “Heavy” (H; Arg4/Lys8)-labeled ethanol-stimulated tachyzoites or "Light" (L; Arg0/Lys0)-labeled non-stimulated parasites was generated, and a TiO 2 -enriched phosphopeptide sample of H/L-labeled Toxoplasma invasion motor complexes was prepared and analysed by LC-MS/MS on an LTQ-Orbitrap instrument. Mascot and MaxQuant search engines facilitated subsequent manual identification, phosphosite localization and quantification of proteins or peptides as detailed in materials and mathods. B ) Sypro Ruby-stained SDS-PAGE separation of the relative amounts of light (lane 1) or heavy (lane 2) Triton X-100 whole protein extracts are shown. Intact tachyzoite invasion motor complexes comprising the five major components MyoA, GAP50, GAP45 and MLC1 were precipitated from a 1∶1 H/L mixture by GAP45-specific immuno-affinity chromatography (lane 3).

    Techniques Used: Flow Cytometry, Labeling, Generated, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Staining, SDS Page, Affinity Chromatography

    17) Product Images from "TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics *"

    Article Title: TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M112.017764

    Overlap of phosphopeptides and phosphosites identified from antiphosphotyrosine- and TiO 2 -based phosphopeptides enrichment methods. A–C , Overlap of identified ( A ) phosphoserine (pSer)-, ( B ) phosphothreonine (pThr)- and ( C ) phosphotyrosine (pTyr)-containing peptides from antiphosphotyrosine- and TiO2-based phosphopeptides enrichment methods; D–F , Overlap of identified ( D ) pSer ( B ) pThr and ( C ) pTyr sites from anti-phosphotyrosine- and TiO2-based phosphopeptides enrichment methods.
    Figure Legend Snippet: Overlap of phosphopeptides and phosphosites identified from antiphosphotyrosine- and TiO 2 -based phosphopeptides enrichment methods. A–C , Overlap of identified ( A ) phosphoserine (pSer)-, ( B ) phosphothreonine (pThr)- and ( C ) phosphotyrosine (pTyr)-containing peptides from antiphosphotyrosine- and TiO2-based phosphopeptides enrichment methods; D–F , Overlap of identified ( D ) pSer ( B ) pThr and ( C ) pTyr sites from anti-phosphotyrosine- and TiO2-based phosphopeptides enrichment methods.

    Techniques Used:

    18) Product Images from "A genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis †Electronic supplementary information (ESI) available: Fig. S1–S21; Tables S1–S5, full experimental details and procedures. See DOI: 10.1039/c5sc03059eClick here for additional data file."

    Article Title: A genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis †Electronic supplementary information (ESI) available: Fig. S1–S21; Tables S1–S5, full experimental details and procedures. See DOI: 10.1039/c5sc03059eClick here for additional data file.

    Journal: Chemical Science

    doi: 10.1039/c5sc03059e

    HPLC-UV, LC-ESI-HRMS and MS–MS analysis of P450 deletion mutants from both Tü 3010 and thiolactomycin (TLM) biosynthetic pathways. (A) HPLC trace profiles (UV 238 nm ) of extracts from S. thiolactonus wild-type strain NRRL 15439 and mutants ΔstuD1 and ΔstuD2, the Lentzea sp. wild-type strain ATCC31319 and ΔtlmD1 mutant. Separation was achieved as described in the materials and methods section. Production of Tü 3010 (retention time 8.39 min) was abolished in both P450 ( stuD1 and stuD2 ) mutants, although the ΔstuD2 mutant produced a new UV-absorbing peak (retention time 10.93 min). Thiolactomycin (retention time 27.14 min) production was lost, with no obvious new UV-absorbing peak, upon disruption of tlmD1 . (B) Further LC-ESI-HRMS analysis of the ΔstuD2 intermediate by selective ion monitoring confirmed it as thiotetromycin ( 2 ) (Fig. S20 † ). Asterisk denotes not detected.
    Figure Legend Snippet: HPLC-UV, LC-ESI-HRMS and MS–MS analysis of P450 deletion mutants from both Tü 3010 and thiolactomycin (TLM) biosynthetic pathways. (A) HPLC trace profiles (UV 238 nm ) of extracts from S. thiolactonus wild-type strain NRRL 15439 and mutants ΔstuD1 and ΔstuD2, the Lentzea sp. wild-type strain ATCC31319 and ΔtlmD1 mutant. Separation was achieved as described in the materials and methods section. Production of Tü 3010 (retention time 8.39 min) was abolished in both P450 ( stuD1 and stuD2 ) mutants, although the ΔstuD2 mutant produced a new UV-absorbing peak (retention time 10.93 min). Thiolactomycin (retention time 27.14 min) production was lost, with no obvious new UV-absorbing peak, upon disruption of tlmD1 . (B) Further LC-ESI-HRMS analysis of the ΔstuD2 intermediate by selective ion monitoring confirmed it as thiotetromycin ( 2 ) (Fig. S20 † ). Asterisk denotes not detected.

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry, Mutagenesis, Produced

    19) Product Images from "Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests"

    Article Title: Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests

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

    doi: 10.1073/pnas.1222144110

    GBP3.1 specifically binds pea aphid BBMV. ( A ) Binding of GBP3.1-EGFP (GBP-EGFP; 50 nM) to pea aphid BBMV was out-competed by addition of synthetic GBP3.1 (50 µM). ( B ) GBP3.1 did not bind to BBMV proteins (10 µg) from H. virescens or L
    Figure Legend Snippet: GBP3.1 specifically binds pea aphid BBMV. ( A ) Binding of GBP3.1-EGFP (GBP-EGFP; 50 nM) to pea aphid BBMV was out-competed by addition of synthetic GBP3.1 (50 µM). ( B ) GBP3.1 did not bind to BBMV proteins (10 µg) from H. virescens or L

    Techniques Used: Binding Assay

    Identification of APN as the GBP3.1 receptor. ( A ) A double derivatized synthetic GBP3.1, with a biotin residue at the N terminus and a UV-cross-linking residue (pbenzoyl- l - phenylalanine, BPa) replacing the tyrosine residue in the loop was used for receptor
    Figure Legend Snippet: Identification of APN as the GBP3.1 receptor. ( A ) A double derivatized synthetic GBP3.1, with a biotin residue at the N terminus and a UV-cross-linking residue (pbenzoyl- l - phenylalanine, BPa) replacing the tyrosine residue in the loop was used for receptor

    Techniques Used:

    20) Product Images from "Deciphering preferential interactions within supramolecular protein complexes: the proteasome case"

    Article Title: Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20145497

    Protein correlation profiling (PCP) analysis of glycerol density gradient-separated proteasome complexes PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15–40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section. PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1–6, Rpn1–3, Rpn5, 7–9, 11–13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ 2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ 2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ 2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel). PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1–α7, β1–β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ 2 values of the experimental replicate 2 as a function of the χ 2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ 2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.
    Figure Legend Snippet: Protein correlation profiling (PCP) analysis of glycerol density gradient-separated proteasome complexes PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15–40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section. PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1–6, Rpn1–3, Rpn5, 7–9, 11–13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ 2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ 2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ 2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel). PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1–α7, β1–β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ 2 values of the experimental replicate 2 as a function of the χ 2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ 2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.

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

    Changes in the expression of 20S proteasome catalytic subunits modulate 20S-associated regulators The two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits. Western blots against the immuno- (β1i, β2i, β5i) and standard (β1, β2, β5) catalytic subunits of the 20S proteasome. Calnexin is used as a loading control. Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non-adjacent in the gel. Importantly, the bands were assembled from the same blot. Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome. The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions ( n = 4). Kinetics of IFN-γ treatment on HeLa cells. HeLa cells were stimulated for 0, 24, 48, or 72 h with IFN-γ. Western blots were performed on total cell lysates with antibodies against the β2i, α2, and α5 subunits. IRF-1 was used to control IFN-γ treatment efficiency, and GAPDH was used as a loading control. For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI ( n = 3). Data information: * P
    Figure Legend Snippet: Changes in the expression of 20S proteasome catalytic subunits modulate 20S-associated regulators The two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits. Western blots against the immuno- (β1i, β2i, β5i) and standard (β1, β2, β5) catalytic subunits of the 20S proteasome. Calnexin is used as a loading control. Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non-adjacent in the gel. Importantly, the bands were assembled from the same blot. Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome. The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions ( n = 4). Kinetics of IFN-γ treatment on HeLa cells. HeLa cells were stimulated for 0, 24, 48, or 72 h with IFN-γ. Western blots were performed on total cell lysates with antibodies against the β2i, α2, and α5 subunits. IRF-1 was used to control IFN-γ treatment efficiency, and GAPDH was used as a loading control. For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI ( n = 3). Data information: * P

    Techniques Used: Expressing, Western Blot, Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Protein abundance correlation of affinity-purified complexes analyzed by mass spectrometry analysis applied to the proteasome complexes and their interacting proteins Heat-map representing the correlations (expressed as the R 2 ) between the abundances of 73 known proteasome-interacting proteins (PIPs) and the abundances of 8 reference proteins or protein complexes, PA28γ, β2i (representing the iP20S), PA28αβ, ncP20S (median of α1–α7, β3, β4, β6, and β7 profiles), 19S (median of Rpt1–6, Rpn1–3, 5–14 profiles), PI31, β5 (representing the sP20S), and PA200. For protein complexes, the median PAI of their subunits in each of the 24 AP-MS experiments was used: α1–α7, β3, β4, β6, and β7 subunits for the ncP20S, Rpt1–6, Rpn1–3, 5–14 for the 19S RP, and PA28α and PA28β subunits for the PA28αβ RP. The R 2 values were hierarchically clustered. Three distinct clusters of composition detailed hereafter could be obtained. Cluster 1 (from top to bottom): Rpt3, Rpn13, α2, Rpn7, USP14, hHR23B, α1, β6, β3, α4, α7, Rpn6, Rpn3, Rpt4, Rpn10, Rpn5, Rpt5, Rpn1, Rpn11, Rpt1, Rpn9, Rpn2, Rpn8, α3, α6, β4, Rpt6, PDC6, Rpn12, APEH, Ubiquilin-1, α5, β7, CCT7, CCT4, CCT2, CCT3, CCT5, CCT6A, DNAJA1, HSP90AB1, HSP90AA1, PNP, Rpt2. Cluster 2 (from top to bottom): 14-3-3ζ/δ, CAND1, GSR, UBE3C, β2, ATP5A1, ATP5B, β1, PA200, β5, FBXO7, UCHL5, TXNL1, ECM29, PI31. Cluster 3 (from top to bottom): PA28β, PITH1, β2i, PA28α, PA28γ, β5i, β1i. Principal component analysis (PCA) of the abundances of 73 known PIPs. The circles represent the main clusters observed (iP20S, ncP20S/19S, sP20S and the 20S assembly chaperones). Plot of the R 2 values between the iP20S or the sP20S and 193 protein correlating ( R 2 > 0.8) with the iP20S, the sP20S, or the ncP20S.
    Figure Legend Snippet: Protein abundance correlation of affinity-purified complexes analyzed by mass spectrometry analysis applied to the proteasome complexes and their interacting proteins Heat-map representing the correlations (expressed as the R 2 ) between the abundances of 73 known proteasome-interacting proteins (PIPs) and the abundances of 8 reference proteins or protein complexes, PA28γ, β2i (representing the iP20S), PA28αβ, ncP20S (median of α1–α7, β3, β4, β6, and β7 profiles), 19S (median of Rpt1–6, Rpn1–3, 5–14 profiles), PI31, β5 (representing the sP20S), and PA200. For protein complexes, the median PAI of their subunits in each of the 24 AP-MS experiments was used: α1–α7, β3, β4, β6, and β7 subunits for the ncP20S, Rpt1–6, Rpn1–3, 5–14 for the 19S RP, and PA28α and PA28β subunits for the PA28αβ RP. The R 2 values were hierarchically clustered. Three distinct clusters of composition detailed hereafter could be obtained. Cluster 1 (from top to bottom): Rpt3, Rpn13, α2, Rpn7, USP14, hHR23B, α1, β6, β3, α4, α7, Rpn6, Rpn3, Rpt4, Rpn10, Rpn5, Rpt5, Rpn1, Rpn11, Rpt1, Rpn9, Rpn2, Rpn8, α3, α6, β4, Rpt6, PDC6, Rpn12, APEH, Ubiquilin-1, α5, β7, CCT7, CCT4, CCT2, CCT3, CCT5, CCT6A, DNAJA1, HSP90AB1, HSP90AA1, PNP, Rpt2. Cluster 2 (from top to bottom): 14-3-3ζ/δ, CAND1, GSR, UBE3C, β2, ATP5A1, ATP5B, β1, PA200, β5, FBXO7, UCHL5, TXNL1, ECM29, PI31. Cluster 3 (from top to bottom): PA28β, PITH1, β2i, PA28α, PA28γ, β5i, β1i. Principal component analysis (PCA) of the abundances of 73 known PIPs. The circles represent the main clusters observed (iP20S, ncP20S/19S, sP20S and the 20S assembly chaperones). Plot of the R 2 values between the iP20S or the sP20S and 193 protein correlating ( R 2 > 0.8) with the iP20S, the sP20S, or the ncP20S.

    Techniques Used: Affinity Purification, Mass Spectrometry

    21) Product Images from "Identification of 3-Sulfinopropionyl Coenzyme A (CoA) Desulfinases within the Acyl-CoA Dehydrogenase Superfamily"

    Article Title: Identification of 3-Sulfinopropionyl Coenzyme A (CoA) Desulfinases within the Acyl-CoA Dehydrogenase Superfamily

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01265-13

    Typical mass spectra of butyryl-CoA and butenoyl-CoA. A sample of the dehydrogenase assay mixture with Acd B4 was subjected to HPLC-ESI-MS analysis to verify the presence of the substrate CoA thioester and the formation of the expected dehydrogenated CoA
    Figure Legend Snippet: Typical mass spectra of butyryl-CoA and butenoyl-CoA. A sample of the dehydrogenase assay mixture with Acd B4 was subjected to HPLC-ESI-MS analysis to verify the presence of the substrate CoA thioester and the formation of the expected dehydrogenated CoA

    Techniques Used: Dehydrogenase Assay, High Performance Liquid Chromatography, Mass Spectrometry

    22) Product Images from "Physicochemical and biological evaluation of JR-131 as a biosimilar to a long-acting erythropoiesis-stimulating agent darbepoetin alfa"

    Article Title: Physicochemical and biological evaluation of JR-131 as a biosimilar to a long-acting erythropoiesis-stimulating agent darbepoetin alfa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0231830

    Total ion chromatograms of trypsin/endoproteinase Glu-C-digested JR-131 (upper) and darbepoetin alfa (lower). Fractions from retention times 11.5 to 13.5 min (T1), 17.0 to 19.5 min (T2), and 24.0 to 26.5 min (T3) were subjected to mass spectrometric profiling ( S2A Fig for T1, S2B Fig for T2, and S2C Fig for T3).
    Figure Legend Snippet: Total ion chromatograms of trypsin/endoproteinase Glu-C-digested JR-131 (upper) and darbepoetin alfa (lower). Fractions from retention times 11.5 to 13.5 min (T1), 17.0 to 19.5 min (T2), and 24.0 to 26.5 min (T3) were subjected to mass spectrometric profiling ( S2A Fig for T1, S2B Fig for T2, and S2C Fig for T3).

    Techniques Used:

    23) Product Images from "Characterisation of the dynamic nature of lipids throughout the lifespan of genetically identical female and male Daphnia magna"

    Article Title: Characterisation of the dynamic nature of lipids throughout the lifespan of genetically identical female and male Daphnia magna

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-62476-z

    Principal component analysis of normalised positive and negative ionisation mode lipidomics data for male and female Daphnia magna demonstrated as percentage of lifespan. Principal component 1 shows clear separation based on sex (F = female, M = male) with females grouped to the left and males to the right. Principal component 2 shows separation by age depicted by colour ranging from orange (young) to blue (old). Female D. magna age range is 10 days to 80 days (maximum sampled age = 80 days). For female age groups 20, 40 and 80 days, eight technical replicates were used and for intermediate ages two technical replicates were used. Male D. magna age groups ranging from 8 days to 40 days were used (maximum sampled age = 40 days). For male D. magna age groups 10, 20 and 40 days, eight technical replicates were used and for intermediate age groups two technical replicates were used. For detailed information regarding biological replicate numbers see Supplementary Tables S1 and S2 . Supplementary Figures S5 and S6 show PCA plots for positive and negative ion mode separately and highlight the grouping of the intrastudy QC sample.
    Figure Legend Snippet: Principal component analysis of normalised positive and negative ionisation mode lipidomics data for male and female Daphnia magna demonstrated as percentage of lifespan. Principal component 1 shows clear separation based on sex (F = female, M = male) with females grouped to the left and males to the right. Principal component 2 shows separation by age depicted by colour ranging from orange (young) to blue (old). Female D. magna age range is 10 days to 80 days (maximum sampled age = 80 days). For female age groups 20, 40 and 80 days, eight technical replicates were used and for intermediate ages two technical replicates were used. Male D. magna age groups ranging from 8 days to 40 days were used (maximum sampled age = 40 days). For male D. magna age groups 10, 20 and 40 days, eight technical replicates were used and for intermediate age groups two technical replicates were used. For detailed information regarding biological replicate numbers see Supplementary Tables S1 and S2 . Supplementary Figures S5 and S6 show PCA plots for positive and negative ion mode separately and highlight the grouping of the intrastudy QC sample.

    Techniques Used:

    Heatmap of lipid changes with sex:age interaction. Heatmap of lipidomics analysis for top 50 lipids significantly-changing concentrations in positive ion mode (dark purple) and top 50 lipids significantly changing levels in negative ion mode (light purple, adjusted p -value
    Figure Legend Snippet: Heatmap of lipid changes with sex:age interaction. Heatmap of lipidomics analysis for top 50 lipids significantly-changing concentrations in positive ion mode (dark purple) and top 50 lipids significantly changing levels in negative ion mode (light purple, adjusted p -value

    Techniques Used:

    24) Product Images from "The Use of Natural Agents to Counteract Telomere Shortening: Effects of a Multi-Component Extract of Astragalus mongholicus Bunge and Danazol"

    Article Title: The Use of Natural Agents to Counteract Telomere Shortening: Effects of a Multi-Component Extract of Astragalus mongholicus Bunge and Danazol

    Journal: Biomedicines

    doi: 10.3390/biomedicines8020031

    High-performance thin-layer chromatography (HPTLC) plate for amino acids, astragalosides and flavonoids. Track 1: A. mongholicus Bunge HRE (0.2 µL), Track 2: astragaloside IV (2 µL), Track 3: A. mongholicus Bunge HRE (1 µL), Track 4: formononetin (4 µL) and Track 5: A. mongholicus Bunge HRE (3.4 µL).
    Figure Legend Snippet: High-performance thin-layer chromatography (HPTLC) plate for amino acids, astragalosides and flavonoids. Track 1: A. mongholicus Bunge HRE (0.2 µL), Track 2: astragaloside IV (2 µL), Track 3: A. mongholicus Bunge HRE (1 µL), Track 4: formononetin (4 µL) and Track 5: A. mongholicus Bunge HRE (3.4 µL).

    Techniques Used: High Performance Thin Layer Chromatography

    25) Product Images from "Analysis of circulating protein aggregates reveals pathological hallmarks of amyotrophic lateral sclerosis"

    Article Title: Analysis of circulating protein aggregates reveals pathological hallmarks of amyotrophic lateral sclerosis

    Journal: bioRxiv

    doi: 10.1101/2020.04.30.070979

    Proteins identified by LC-MS/MS in circulating protein aggregates (CPA) enriched from ALS and HC pooled plasma samples and from aggregates enriched from brain. (A) Venn diagram showing CPA proteins unique to or shared by ALS and HC. (B) Venn diagram showing HC and ALS CPA proteins shared by brain aggregates. Five proteins were expressed in all 3 aggregate groups (actin cytoplasmic 1, tubulin alpha-4A chain isoform 2, clathrin heavy chain 1 isoform 2, collagen alpha-1(VI) and plectin isoform 7), while brain aggregates shared only one protein with ALS and HC CPA (cytoplasmic dynein 1 heavy chain 1 and collagen alpha-2(VI), respectively).
    Figure Legend Snippet: Proteins identified by LC-MS/MS in circulating protein aggregates (CPA) enriched from ALS and HC pooled plasma samples and from aggregates enriched from brain. (A) Venn diagram showing CPA proteins unique to or shared by ALS and HC. (B) Venn diagram showing HC and ALS CPA proteins shared by brain aggregates. Five proteins were expressed in all 3 aggregate groups (actin cytoplasmic 1, tubulin alpha-4A chain isoform 2, clathrin heavy chain 1 isoform 2, collagen alpha-1(VI) and plectin isoform 7), while brain aggregates shared only one protein with ALS and HC CPA (cytoplasmic dynein 1 heavy chain 1 and collagen alpha-2(VI), respectively).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    TMTcalibrator™ experimental design. (A) Tandem Mass Tag (TMT) reagents with relative masses and isotope position. (B) General 10plex labelling layout after trypsin digestion of the samples; analytical samples and calilbrants are mixed in a specific ratio that enhances detection by LC-MS/MS of low abundant peptides in the analytical channels thanks to the high calibrant content. (C) Labelling strategy in two 10plexes LC-MS/MS runs which includes Circulating Protein aggregates (CPA) from amyotrophic lateral sclerosis (ALS) patients and from healthy controls (HC) in the analytical channels (orange and blue colour codes) and a mixture (1:1) of brains (Precentral gyrus) lysates from two different ALS patients in the calibrant channels (grey colour code).
    Figure Legend Snippet: TMTcalibrator™ experimental design. (A) Tandem Mass Tag (TMT) reagents with relative masses and isotope position. (B) General 10plex labelling layout after trypsin digestion of the samples; analytical samples and calilbrants are mixed in a specific ratio that enhances detection by LC-MS/MS of low abundant peptides in the analytical channels thanks to the high calibrant content. (C) Labelling strategy in two 10plexes LC-MS/MS runs which includes Circulating Protein aggregates (CPA) from amyotrophic lateral sclerosis (ALS) patients and from healthy controls (HC) in the analytical channels (orange and blue colour codes) and a mixture (1:1) of brains (Precentral gyrus) lysates from two different ALS patients in the calibrant channels (grey colour code).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    TMTcalibrator™: bioinformatic pipeline. After MS/MS spectra (raw data) acquisition, the intensity of each channel was corrected for background and cross-talking between tags in the second mass spectrometer (MS2). Intensity values of the detected Peptide-Spectrum Matches (PSMs) were normalised with a reference value generated as the average of the Calibrant channels and this was followed by PSM-to-peptide summarisation defined as “trimmed mean”. In this passage the data points considered as outliers in each analytical sample were removed stabilizing the mean before merging the data obtained from the two 10plexes analysed. Then, “not available data points (NA)” filtering, imputation and quantile normalization were performed on the merged data set, so that it was possible to perform a Principal Component Analysis (PCA) on the data acquired. It was also possible to evaluate the TMT batch effect within linear models for microarray data (LIMMA). After peptide-to-protein summarisation, statistical significance of the protein groups identified was analysed with LIMMA-based t-test (p-Value
    Figure Legend Snippet: TMTcalibrator™: bioinformatic pipeline. After MS/MS spectra (raw data) acquisition, the intensity of each channel was corrected for background and cross-talking between tags in the second mass spectrometer (MS2). Intensity values of the detected Peptide-Spectrum Matches (PSMs) were normalised with a reference value generated as the average of the Calibrant channels and this was followed by PSM-to-peptide summarisation defined as “trimmed mean”. In this passage the data points considered as outliers in each analytical sample were removed stabilizing the mean before merging the data obtained from the two 10plexes analysed. Then, “not available data points (NA)” filtering, imputation and quantile normalization were performed on the merged data set, so that it was possible to perform a Principal Component Analysis (PCA) on the data acquired. It was also possible to evaluate the TMT batch effect within linear models for microarray data (LIMMA). After peptide-to-protein summarisation, statistical significance of the protein groups identified was analysed with LIMMA-based t-test (p-Value

    Techniques Used: Tandem Mass Spectroscopy, Mass Spectrometry, Generated, Analytical Sample Preparation, Microarray, Significance Assay

    Comparison of aggregation propensity parameters of CPA proteins obtained through the TMT proteomic workflow (TMT-CPA), BPA proteins by LC-MS/MS and the Human proteome. Molecular weight (MW) (A), isoelectric point (pI) (B) and hydrophobicity (GRAVY index) (C) were compared across TMT-CPA, BPA and Human proteome datasets. Statistical analysis was performed with one-way ANOVA, Kruskal-Wallis test with Dunn’s multiple comparison as post-test. The violin plots show median and interquartile ranges across the three datasets. The TMT-CPA dataset differ significantly compared to the Human proteome and BPA for MW and pI, while for hydrophobicity (GRAVY index), there is only a less significant difference with the BPA dataset. **** p
    Figure Legend Snippet: Comparison of aggregation propensity parameters of CPA proteins obtained through the TMT proteomic workflow (TMT-CPA), BPA proteins by LC-MS/MS and the Human proteome. Molecular weight (MW) (A), isoelectric point (pI) (B) and hydrophobicity (GRAVY index) (C) were compared across TMT-CPA, BPA and Human proteome datasets. Statistical analysis was performed with one-way ANOVA, Kruskal-Wallis test with Dunn’s multiple comparison as post-test. The violin plots show median and interquartile ranges across the three datasets. The TMT-CPA dataset differ significantly compared to the Human proteome and BPA for MW and pI, while for hydrophobicity (GRAVY index), there is only a less significant difference with the BPA dataset. **** p

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Molecular Weight

    26) Product Images from "NprR-NprX Quorum-Sensing System Regulates the Algicidal Activity of Bacillus sp. Strain S51107 against Bloom-Forming Cyanobacterium Microcystis aeruginosa"

    Article Title: NprR-NprX Quorum-Sensing System Regulates the Algicidal Activity of Bacillus sp. Strain S51107 against Bloom-Forming Cyanobacterium Microcystis aeruginosa

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.01968

    Agar plate assay and MS/MS analysis of the NprX peptide. (A) Paper disk diffusion assay of NprX peptide extracted by a Waters Sep Pak C18 cartridge by using the reporter strain Bacillus thuringiensis 407 Cry - ( nprA′Z ΔRX [pHT304-R]). The left plate is an extract of BEP medium (control), and the right plate is an extract of the culture supernatant from strain S51107. (B) MS/MS spectra fragmentation of the SKPDIVG synthetic heptapeptide (standard substance). (C) MS/MS spectra fragmentation of NprX peptide extracts from strain S51107 culture. (D) Theoretical fragmentation of the SKPDIVG peptide.
    Figure Legend Snippet: Agar plate assay and MS/MS analysis of the NprX peptide. (A) Paper disk diffusion assay of NprX peptide extracted by a Waters Sep Pak C18 cartridge by using the reporter strain Bacillus thuringiensis 407 Cry - ( nprA′Z ΔRX [pHT304-R]). The left plate is an extract of BEP medium (control), and the right plate is an extract of the culture supernatant from strain S51107. (B) MS/MS spectra fragmentation of the SKPDIVG synthetic heptapeptide (standard substance). (C) MS/MS spectra fragmentation of NprX peptide extracts from strain S51107 culture. (D) Theoretical fragmentation of the SKPDIVG peptide.

    Techniques Used: Mass Spectrometry, Diffusion-based Assay

    TEM observations of the degradation process of M. aeruginosa treated with Bacillus sp. strain S51107. (A) Normal cell; (B) damaged cell wall; (C) ruptured cell wall and membrane; (D) distorted and broken cell. CW, cell wall; CM, cell membrane; Th, thylakoids; Ld, lipid droplets; Cg, cyanophycin granules; Pg, polyphosphate granules; BC, bacterial cells; MA, M. aeruginosa . The arrows in (B) show damaged (up) and intact (down) cell walls. The arrows in (C) indicate the partly ruptured M. aeruginosa cell.
    Figure Legend Snippet: TEM observations of the degradation process of M. aeruginosa treated with Bacillus sp. strain S51107. (A) Normal cell; (B) damaged cell wall; (C) ruptured cell wall and membrane; (D) distorted and broken cell. CW, cell wall; CM, cell membrane; Th, thylakoids; Ld, lipid droplets; Cg, cyanophycin granules; Pg, polyphosphate granules; BC, bacterial cells; MA, M. aeruginosa . The arrows in (B) show damaged (up) and intact (down) cell walls. The arrows in (C) indicate the partly ruptured M. aeruginosa cell.

    Techniques Used: Transmission Electron Microscopy

    Algicidal effects of silica gel chromatography fractions on cyanobacterial-lawn and high performance liquid chromatography (HPLC) separation of the algicidal fractions S2 and S5. (A–C) indicate the cyanobacterial-lawn results of algicidal fraction S2, S5 and control (other fractions), respectively. HPLC was performed on a Zorbax ® Bonus-RP column (4.6 mm × 250 mm, 5 μm). The fraction S2 was eluted with a linear gradient of MeOH/H 2 O from 5 to 47% (vol/vol) for 60 min at a flow rate of 1.0 ml/min, yielding pure S51107-A (retention time = 47.5–49.0 min) (D) . The fraction S5 was purified with MeOH/H 2 O from 10 to 60% for 40 min yielded pure S51107-B (retention time = 22.6–23.2 min) (E) . The arrows denote the algicidal effect of fraction on the cyanobacterial-lawn.
    Figure Legend Snippet: Algicidal effects of silica gel chromatography fractions on cyanobacterial-lawn and high performance liquid chromatography (HPLC) separation of the algicidal fractions S2 and S5. (A–C) indicate the cyanobacterial-lawn results of algicidal fraction S2, S5 and control (other fractions), respectively. HPLC was performed on a Zorbax ® Bonus-RP column (4.6 mm × 250 mm, 5 μm). The fraction S2 was eluted with a linear gradient of MeOH/H 2 O from 5 to 47% (vol/vol) for 60 min at a flow rate of 1.0 ml/min, yielding pure S51107-A (retention time = 47.5–49.0 min) (D) . The fraction S5 was purified with MeOH/H 2 O from 10 to 60% for 40 min yielded pure S51107-B (retention time = 22.6–23.2 min) (E) . The arrows denote the algicidal effect of fraction on the cyanobacterial-lawn.

    Techniques Used: Chromatography, High Performance Liquid Chromatography, Flow Cytometry, Purification

    Dynamics of the cell densities of M. aeruginosa 9110 (A) and the algicidal strains (B) and the concentration of dissolved organic carbon (DOC) (C) during the algicidal process of Bacillus sp. strain S51107 and its mutants against M. aeruginosa 9110. The data are shown as the averages of three independent experiments (error bars are the SD from the mean values). The values of group B marked with ∗ were significantly different ( P
    Figure Legend Snippet: Dynamics of the cell densities of M. aeruginosa 9110 (A) and the algicidal strains (B) and the concentration of dissolved organic carbon (DOC) (C) during the algicidal process of Bacillus sp. strain S51107 and its mutants against M. aeruginosa 9110. The data are shown as the averages of three independent experiments (error bars are the SD from the mean values). The values of group B marked with ∗ were significantly different ( P

    Techniques Used: Concentration Assay

    Algicidal activities of different treatments of Bacillus sp. strain S51107 cultures against Microcystis aeruginosa 9110 after 6 days of inoculation. The data are the averages of three independent experiments (error bars are the SD from mean values). Different letters indicate statistically significant differences ( P
    Figure Legend Snippet: Algicidal activities of different treatments of Bacillus sp. strain S51107 cultures against Microcystis aeruginosa 9110 after 6 days of inoculation. The data are the averages of three independent experiments (error bars are the SD from mean values). Different letters indicate statistically significant differences ( P

    Techniques Used:

    27) Product Images from "Bis(monoacylglycero)phosphate lipids in the retinal pigment epithelium implicate lysosomal/endosomal dysfunction in a model of Stargardt disease and human retinas"

    Article Title: Bis(monoacylglycero)phosphate lipids in the retinal pigment epithelium implicate lysosomal/endosomal dysfunction in a model of Stargardt disease and human retinas

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17402-1

    LC-MS data from Abca4 −/− Folch extracted retina homogenate with corresponding structures. ( a ) Extracted ion chromatogram displaying the retention time of m/z 865.50. ( b ) Full scan orbitrap MS spectrum in negative ion mode from peak displayed in ( a ) showing m/z 865.50 as the base peak. ( c ) MS/MS mass spectrum in negative ion mode from the mass selected [M-H] − ion at m/z 865.50. ( d ) MS/MS mass spectrum in positive ion mode from the mass selected [M + Li] + adduct at m/z 873.5.
    Figure Legend Snippet: LC-MS data from Abca4 −/− Folch extracted retina homogenate with corresponding structures. ( a ) Extracted ion chromatogram displaying the retention time of m/z 865.50. ( b ) Full scan orbitrap MS spectrum in negative ion mode from peak displayed in ( a ) showing m/z 865.50 as the base peak. ( c ) MS/MS mass spectrum in negative ion mode from the mass selected [M-H] − ion at m/z 865.50. ( d ) MS/MS mass spectrum in positive ion mode from the mass selected [M + Li] + adduct at m/z 873.5.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    28) Product Images from "Characterization of human enterovirus71 virus-like particles used for vaccine antigens"

    Article Title: Characterization of human enterovirus71 virus-like particles used for vaccine antigens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181182

    Confirmation of amino acid sequence of EV71 VLPs. Amino acid sequence of VP0 (a), VP1 (b) and VP3 (c) of EV71 VLPs (A), as determined by translation of the corresponding DNA sequences. Peptides are shown that have been obtained by digestion with trypsin (blue), chymotrypsin (green) or endoproteinase Glu-C (red) and identified by LC-MS/MS. The N-terminal sequences are marked in orange and the C-terminal sequences in purple. (B) N-glycosylation site at VP1 residue N 176 of EV71 VLPs. Monoisotopic mass of neutral peptide Mr (calc): 1777.8887; fixed modifications: Carbamidomethyl (C) (applied to specified residues or termini only); variable modifications: N10: deamidated (NQ); ions score: 57 Expect: 2.1e-006; matches: 21/162 fragment ions using 39 of the most intense peaks. Electrospray MS/MS spectra were assigned to the EV71 VLPs primary sequence using the Mascot 2.1.0 (Matrix Science, London, UK) software, and an in-house protein sequence database was established.
    Figure Legend Snippet: Confirmation of amino acid sequence of EV71 VLPs. Amino acid sequence of VP0 (a), VP1 (b) and VP3 (c) of EV71 VLPs (A), as determined by translation of the corresponding DNA sequences. Peptides are shown that have been obtained by digestion with trypsin (blue), chymotrypsin (green) or endoproteinase Glu-C (red) and identified by LC-MS/MS. The N-terminal sequences are marked in orange and the C-terminal sequences in purple. (B) N-glycosylation site at VP1 residue N 176 of EV71 VLPs. Monoisotopic mass of neutral peptide Mr (calc): 1777.8887; fixed modifications: Carbamidomethyl (C) (applied to specified residues or termini only); variable modifications: N10: deamidated (NQ); ions score: 57 Expect: 2.1e-006; matches: 21/162 fragment ions using 39 of the most intense peaks. Electrospray MS/MS spectra were assigned to the EV71 VLPs primary sequence using the Mascot 2.1.0 (Matrix Science, London, UK) software, and an in-house protein sequence database was established.

    Techniques Used: Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Software

    29) Product Images from "Infection-stage adjusted dose of beta-lactams for parsimonious and efficient antibiotic treatments: A Pasteurella multocida experimental pneumonia in mice"

    Article Title: Infection-stage adjusted dose of beta-lactams for parsimonious and efficient antibiotic treatments: A Pasteurella multocida experimental pneumonia in mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0182863

    Amoxicillin and cefquinome pharmacokinetics in mice. Observed (○) and predicted (-) plasma antibiotic concentrations versus time in mice (n = 3 at each time point) after a single subcutaneous administration. A) Amoxicillin, 50 mg/kg. B) Cefquinome, 5 mg/kg. Cefquinome concentrations were below the LOQ for one mouse at 4 h, 2 mice at 6, 8 and 24 h and 3 mice at 12 h.
    Figure Legend Snippet: Amoxicillin and cefquinome pharmacokinetics in mice. Observed (○) and predicted (-) plasma antibiotic concentrations versus time in mice (n = 3 at each time point) after a single subcutaneous administration. A) Amoxicillin, 50 mg/kg. B) Cefquinome, 5 mg/kg. Cefquinome concentrations were below the LOQ for one mouse at 4 h, 2 mice at 6, 8 and 24 h and 3 mice at 12 h.

    Techniques Used: Mouse Assay

    Time-course of the administered treatments. Mice were infected at Day 0 and were treated or not with amoxicillin or cefquinome depending on the treatment phase (control, pre-patent or patent treatment) and on the group. Mice were sacrificed seven days after the challenge or the beginning of treatment (triangle). Arrows represent the administrations of amoxicillin or cefquinome (each treatment corresponded to 4 daily administrations). The administered doses depended on the treatment phase (control, pre-patent or patent treatment) and on the group. The star indicates that at least one clinical symptom was observed and that the mouse was considered as sick.
    Figure Legend Snippet: Time-course of the administered treatments. Mice were infected at Day 0 and were treated or not with amoxicillin or cefquinome depending on the treatment phase (control, pre-patent or patent treatment) and on the group. Mice were sacrificed seven days after the challenge or the beginning of treatment (triangle). Arrows represent the administrations of amoxicillin or cefquinome (each treatment corresponded to 4 daily administrations). The administered doses depended on the treatment phase (control, pre-patent or patent treatment) and on the group. The star indicates that at least one clinical symptom was observed and that the mouse was considered as sick.

    Techniques Used: Mouse Assay, Infection

    Clinical and microbiological cure rates with amoxicillin. Clinical (dark bars) and microbiological (light bars) cure rates of mice after no treatment (grey/black), early (green) or late (red) treatments with different doses of amoxicillin (2.5, 5, 25, or 50 mg/kg). Rates were calculated for all the mice of the group in control and early treatments (as all the mice in a given group were subjected to the same protocol) whereas cure rates for the late treatments were only calculated from the 60 to 70% of mice treated with amoxicillin NA: not assessed.
    Figure Legend Snippet: Clinical and microbiological cure rates with amoxicillin. Clinical (dark bars) and microbiological (light bars) cure rates of mice after no treatment (grey/black), early (green) or late (red) treatments with different doses of amoxicillin (2.5, 5, 25, or 50 mg/kg). Rates were calculated for all the mice of the group in control and early treatments (as all the mice in a given group were subjected to the same protocol) whereas cure rates for the late treatments were only calculated from the 60 to 70% of mice treated with amoxicillin NA: not assessed.

    Techniques Used: Mouse Assay

    30) Product Images from "Formation and determination of the oxidation products of 5-methylcytosine in RNA and determination of the oxidation products of 5-methylcytosine in RNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01589a"

    Article Title: Formation and determination of the oxidation products of 5-methylcytosine in RNA and determination of the oxidation products of 5-methylcytosine in RNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01589a

    Journal: Chemical Science

    doi: 10.1039/c6sc01589a

    Confirmation of the cytosine modifications in the RNA of mammals using high-resolution mass spectrometry. (A) Product-ion spectra of BDEPE labeled 5-mC standard (left) and 5-mC from the RNA of human CRC tissue. (B) Product-ion spectra of BDEPE labeled 5-hmC standard (left) and 5-hmC from the RNA of human CRC tissue. (C) Product-ion spectra of BDEPE labeled 5-foC standard (left) and 5-foC from the RNA of human CRC tissue. (D) Product-ion spectra of BDEPE labeled 5-caC standard (left) and 5-caC from the RNA of human CRC tissue.
    Figure Legend Snippet: Confirmation of the cytosine modifications in the RNA of mammals using high-resolution mass spectrometry. (A) Product-ion spectra of BDEPE labeled 5-mC standard (left) and 5-mC from the RNA of human CRC tissue. (B) Product-ion spectra of BDEPE labeled 5-hmC standard (left) and 5-hmC from the RNA of human CRC tissue. (C) Product-ion spectra of BDEPE labeled 5-foC standard (left) and 5-foC from the RNA of human CRC tissue. (D) Product-ion spectra of BDEPE labeled 5-caC standard (left) and 5-caC from the RNA of human CRC tissue.

    Techniques Used: Mass Spectrometry, Labeling

    Quantification and statistical analysis of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the mRNA of human HCC tissues and tumor adjacent normal tissues.
    Figure Legend Snippet: Quantification and statistical analysis of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the mRNA of human HCC tissues and tumor adjacent normal tissues.

    Techniques Used:

    Determination of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in different RNA species from mouse liver tissue. 10 μg total RNA and 1 μg other RNA species were used for quantification.
    Figure Legend Snippet: Determination of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in different RNA species from mouse liver tissue. 10 μg total RNA and 1 μg other RNA species were used for quantification.

    Techniques Used:

    Quantification and statistical analysis of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the total RNA of human CRC tissues and tumor adjacent normal tissues (left panel) or in the total RNA of human HCC tissues and tumor adjacent normal tissues (right panel).
    Figure Legend Snippet: Quantification and statistical analysis of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the total RNA of human CRC tissues and tumor adjacent normal tissues (left panel) or in the total RNA of human HCC tissues and tumor adjacent normal tissues (right panel).

    Techniques Used:

    Product ions spectra of BDEPE labeled 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D).
    Figure Legend Snippet: Product ions spectra of BDEPE labeled 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D).

    Techniques Used: Labeling

    Determination of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the RNA of mammals using BDEPE labeling coupled with LC-ESI-MS/MS analysis. Extracted ion chromatograms of the BDEPE labeled standards, BDEPE labeled cytosine modifications in the RNA from human CRC tissue, mouse liver tissue and 293T cells are shown in each panel.
    Figure Legend Snippet: Determination of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the RNA of mammals using BDEPE labeling coupled with LC-ESI-MS/MS analysis. Extracted ion chromatograms of the BDEPE labeled standards, BDEPE labeled cytosine modifications in the RNA from human CRC tissue, mouse liver tissue and 293T cells are shown in each panel.

    Techniques Used: Labeling, Mass Spectrometry

    Extracted ion chromatograms of 5-mC, 5-hmC, 5-foC, and 5-caC before (A) and after (B) labeling using BDEPE under the optimized conditions. The amount of 5-mC, 5-hmC, 5-foC, and 5-caC were 200 fmol.
    Figure Legend Snippet: Extracted ion chromatograms of 5-mC, 5-hmC, 5-foC, and 5-caC before (A) and after (B) labeling using BDEPE under the optimized conditions. The amount of 5-mC, 5-hmC, 5-foC, and 5-caC were 200 fmol.

    Techniques Used: Labeling

    31) Product Images from "Formation and determination of the oxidation products of 5-methylcytosine in RNA Formation and determination of the oxidation products of 5-methylcytosine in RNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01589a"

    Article Title: Formation and determination of the oxidation products of 5-methylcytosine in RNA Formation and determination of the oxidation products of 5-methylcytosine in RNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01589a

    Journal: Chemical Science

    doi: 10.1039/c6sc01589a

    Confirmation of the cytosine modifications in the RNA of mammals using high-resolution mass spectrometry. (A) Product-ion spectra of BDEPE labeled 5-mC standard (left) and 5-mC from the RNA of human CRC tissue. (B) Product-ion spectra of BDEPE labeled 5-hmC standard (left) and 5-hmC from the RNA of human CRC tissue. (C) Product-ion spectra of BDEPE labeled 5-foC standard (left) and 5-foC from the RNA of human CRC tissue. (D) Product-ion spectra of BDEPE labeled 5-caC standard (left) and 5-caC from the RNA of human CRC tissue.
    Figure Legend Snippet: Confirmation of the cytosine modifications in the RNA of mammals using high-resolution mass spectrometry. (A) Product-ion spectra of BDEPE labeled 5-mC standard (left) and 5-mC from the RNA of human CRC tissue. (B) Product-ion spectra of BDEPE labeled 5-hmC standard (left) and 5-hmC from the RNA of human CRC tissue. (C) Product-ion spectra of BDEPE labeled 5-foC standard (left) and 5-foC from the RNA of human CRC tissue. (D) Product-ion spectra of BDEPE labeled 5-caC standard (left) and 5-caC from the RNA of human CRC tissue.

    Techniques Used: Mass Spectrometry, Labeling

    Quantification and statistical analysis of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the mRNA of human HCC tissues and tumor adjacent normal tissues.
    Figure Legend Snippet: Quantification and statistical analysis of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the mRNA of human HCC tissues and tumor adjacent normal tissues.

    Techniques Used:

    Determination of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in different RNA species from mouse liver tissue. 10 μg total RNA and 1 μg other RNA species were used for quantification.
    Figure Legend Snippet: Determination of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in different RNA species from mouse liver tissue. 10 μg total RNA and 1 μg other RNA species were used for quantification.

    Techniques Used:

    Quantification and statistical analysis of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the total RNA of human CRC tissues and tumor adjacent normal tissues (left panel) or in the total RNA of human HCC tissues and tumor adjacent normal tissues (right panel).
    Figure Legend Snippet: Quantification and statistical analysis of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the total RNA of human CRC tissues and tumor adjacent normal tissues (left panel) or in the total RNA of human HCC tissues and tumor adjacent normal tissues (right panel).

    Techniques Used:

    Product ions spectra of BDEPE labeled 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D).
    Figure Legend Snippet: Product ions spectra of BDEPE labeled 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D).

    Techniques Used: Labeling

    Determination of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the RNA of mammals using BDEPE labeling coupled with LC-ESI-MS/MS analysis. Extracted ion chromatograms of the BDEPE labeled standards, BDEPE labeled cytosine modifications in the RNA from human CRC tissue, mouse liver tissue and 293T cells are shown in each panel.
    Figure Legend Snippet: Determination of 5-mC (A), 5-hmC (B), 5-foC (C), and 5-caC (D) in the RNA of mammals using BDEPE labeling coupled with LC-ESI-MS/MS analysis. Extracted ion chromatograms of the BDEPE labeled standards, BDEPE labeled cytosine modifications in the RNA from human CRC tissue, mouse liver tissue and 293T cells are shown in each panel.

    Techniques Used: Labeling, Mass Spectrometry

    Extracted ion chromatograms of 5-mC, 5-hmC, 5-foC, and 5-caC before (A) and after (B) labeling using BDEPE under the optimized conditions. The amount of 5-mC, 5-hmC, 5-foC, and 5-caC were 200 fmol.
    Figure Legend Snippet: Extracted ion chromatograms of 5-mC, 5-hmC, 5-foC, and 5-caC before (A) and after (B) labeling using BDEPE under the optimized conditions. The amount of 5-mC, 5-hmC, 5-foC, and 5-caC were 200 fmol.

    Techniques Used: Labeling

    32) Product Images from "Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts"

    Article Title: Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

    Journal: Clinical Proteomics

    doi: 10.1186/s12014-018-9197-x

    Phosphotyrosine profiling of cancer epithelial cells and interacting CAFs. a , b Density scatter plot of log 2 transformed phosphopeptide intensity ratios (82T-co-cultured vs. 82T (A) and MDA-MB-231-co-cultured vs. MDA-MB-231) from two SILAC biological experiments. c Pie chart showing the composition of pTyr and pSer/Thr peptides identified in the phosphoproteomic analysis. d Venn diagram showing overlap of phosphopeptides identified in MDA-MB-231 and 82T cells. e , f Gene ontology analysis of phosphoproteins in cancer epithelium and CAFs. e Cellular component; f molecular functions
    Figure Legend Snippet: Phosphotyrosine profiling of cancer epithelial cells and interacting CAFs. a , b Density scatter plot of log 2 transformed phosphopeptide intensity ratios (82T-co-cultured vs. 82T (A) and MDA-MB-231-co-cultured vs. MDA-MB-231) from two SILAC biological experiments. c Pie chart showing the composition of pTyr and pSer/Thr peptides identified in the phosphoproteomic analysis. d Venn diagram showing overlap of phosphopeptides identified in MDA-MB-231 and 82T cells. e , f Gene ontology analysis of phosphoproteins in cancer epithelium and CAFs. e Cellular component; f molecular functions

    Techniques Used: Transformation Assay, Cell Culture, Multiple Displacement Amplification

    33) Product Images from "Identification of an analgesic lipopeptide produced by the probiotic Escherichia coli strain Nissle 1917"

    Article Title: Identification of an analgesic lipopeptide produced by the probiotic Escherichia coli strain Nissle 1917

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01403-9

    Characterization of C12-Asparagine-aminobutyric acid by LC-HRMS. a EIC of a lipidic extract of EcNwt (up) and EcNΔclbA pellet (down) for m / z 398.2664. No signal was detected in the mutated strain. b Natural isotopic distribution of the deprotonated molecule displayed by the high-resolution mass spectrum zoom obtained for the peak eluted 15.33 min in both TIC (top) at 12.96 min in the probiotic strain EIC (top) and natural isotopic pattern calculated with the formula [(C 20 H 37 N 3 O 5 )-H] − . Analogous natural isotopic patterns and similar m / z ratios measured and simulated for the monoisotopic [( 12 C 20 1 H 37 14 N 3 16 O 5 )-H] − ion and for the [( 13 C n C 20-n 1 H 37 14 N 3 16 O 5 )-H] − ] − (with n = 1 and 2) ions (within an accuracy of 1.8 ppm). Same isotopic profile and a mass accuracy of 1.8 ppm were obtained. c Product ion spectrum acquired via HCD (NCE = 35%) of the carboxylate anion [M-H] − ( m / z 398) generated in electrospray from the LC peak eluted at 12.96 min. d Upper panel: chromatogram obtained for the four synthesized standards: C12-Asn-γ-aminobutyric acid (C12Asn-GABA OH ), C12Asn-( S )AABA OH and C12Asn-( R )AABA OH , C12-Asn-α-aminobutyric acid (C12Asn-BABA OH ) and C12-Asn-α-aminobutyric acid (C12Asn-AABA OH ) (two diastereoisomers are detected which present similar HCD spectrum with NCE = 35%); lower panel: Chromatogram obtained for the lipid extract of EcNwt pellet
    Figure Legend Snippet: Characterization of C12-Asparagine-aminobutyric acid by LC-HRMS. a EIC of a lipidic extract of EcNwt (up) and EcNΔclbA pellet (down) for m / z 398.2664. No signal was detected in the mutated strain. b Natural isotopic distribution of the deprotonated molecule displayed by the high-resolution mass spectrum zoom obtained for the peak eluted 15.33 min in both TIC (top) at 12.96 min in the probiotic strain EIC (top) and natural isotopic pattern calculated with the formula [(C 20 H 37 N 3 O 5 )-H] − . Analogous natural isotopic patterns and similar m / z ratios measured and simulated for the monoisotopic [( 12 C 20 1 H 37 14 N 3 16 O 5 )-H] − ion and for the [( 13 C n C 20-n 1 H 37 14 N 3 16 O 5 )-H] − ] − (with n = 1 and 2) ions (within an accuracy of 1.8 ppm). Same isotopic profile and a mass accuracy of 1.8 ppm were obtained. c Product ion spectrum acquired via HCD (NCE = 35%) of the carboxylate anion [M-H] − ( m / z 398) generated in electrospray from the LC peak eluted at 12.96 min. d Upper panel: chromatogram obtained for the four synthesized standards: C12-Asn-γ-aminobutyric acid (C12Asn-GABA OH ), C12Asn-( S )AABA OH and C12Asn-( R )AABA OH , C12-Asn-α-aminobutyric acid (C12Asn-BABA OH ) and C12-Asn-α-aminobutyric acid (C12Asn-AABA OH ) (two diastereoisomers are detected which present similar HCD spectrum with NCE = 35%); lower panel: Chromatogram obtained for the lipid extract of EcNwt pellet

    Techniques Used: Generated, Synthesized

    34) Product Images from "The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase"

    Article Title: The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase

    Journal:

    doi: 10.1073/pnas.0504996102

    Overall structure of BTK-AiiA and structural comparison of various zinc-metalloenzymes. ( A ) Ribbon representation of BTK-AiiA. α helices and β sheets are shown in purple and green, respectively. The flexible fragment of disordered electron
    Figure Legend Snippet: Overall structure of BTK-AiiA and structural comparison of various zinc-metalloenzymes. ( A ) Ribbon representation of BTK-AiiA. α helices and β sheets are shown in purple and green, respectively. The flexible fragment of disordered electron

    Techniques Used:

    Substrate specificity of BTK-AiiA and inhibition of BTK-AiiA activity by HSL. ( A ) Activity toward C6-AHL (68.6 nmol/min per mg) was defined as 100%. ( B ) A typical steady-state kinetics experiment of BTK-AiiA activity for different C6-AHL concentrations
    Figure Legend Snippet: Substrate specificity of BTK-AiiA and inhibition of BTK-AiiA activity by HSL. ( A ) Activity toward C6-AHL (68.6 nmol/min per mg) was defined as 100%. ( B ) A typical steady-state kinetics experiment of BTK-AiiA activity for different C6-AHL concentrations

    Techniques Used: Inhibition, Activity Assay

    Proposed catalysis mechanism for BTK-AiiA based on the BTK-AiiA–HSL complex and suggested catalysis mechanisms for other zinc metalloenzymes. See text for description.
    Figure Legend Snippet: Proposed catalysis mechanism for BTK-AiiA based on the BTK-AiiA–HSL complex and suggested catalysis mechanisms for other zinc metalloenzymes. See text for description.

    Techniques Used:

    Substrate binding by BTK-AiiA. ( A ) Electron density map showing the bound l -HSL in the active site of BTK-AiiA. The F o – F c map was calculated before the inclusion of l -HSL in the model and is contoured at 2.5 σ. ( B ) HSL-binding site and
    Figure Legend Snippet: Substrate binding by BTK-AiiA. ( A ) Electron density map showing the bound l -HSL in the active site of BTK-AiiA. The F o – F c map was calculated before the inclusion of l -HSL in the model and is contoured at 2.5 σ. ( B ) HSL-binding site and

    Techniques Used: Binding Assay

    35) Product Images from "Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants"

    Article Title: Quantitative Proteomics of the 2016 WHO Neisseria gonorrhoeae Reference Strains Surveys Vaccine Candidates and Antimicrobial Resistance Determinants

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA118.001125

    Experimental paradigm of quantitative proteomic profiling of the N. gonorrhoeae 2016 WHO reference strains and the FA6140 strain. All gonococci were cultured concurrently in liquid medium until reaching mid-logarithmic growth. Bacterial cells were harvested, lysed, and subjected to subcellular fractionation to separate the crude cell envelope (CE) and cytoplasmic (C) proteomes. CE proteins were enriched using a sodium carbonate wash and ultracentrifugation. The obtained CE and C protein samples (100 μg) were denatured, reduced, alkylated, trypsinized, and the peptides from each strain were labeled using 10-plex and 6-plex Tandem mass tag (TMT) reagents, as indicated. Finally, samples were pooled, fractionated by strong cation exchange, and analyzed by liquid chromatography electrospray ionization mass spectrometry. Experiments were performed in biological duplicates.
    Figure Legend Snippet: Experimental paradigm of quantitative proteomic profiling of the N. gonorrhoeae 2016 WHO reference strains and the FA6140 strain. All gonococci were cultured concurrently in liquid medium until reaching mid-logarithmic growth. Bacterial cells were harvested, lysed, and subjected to subcellular fractionation to separate the crude cell envelope (CE) and cytoplasmic (C) proteomes. CE proteins were enriched using a sodium carbonate wash and ultracentrifugation. The obtained CE and C protein samples (100 μg) were denatured, reduced, alkylated, trypsinized, and the peptides from each strain were labeled using 10-plex and 6-plex Tandem mass tag (TMT) reagents, as indicated. Finally, samples were pooled, fractionated by strong cation exchange, and analyzed by liquid chromatography electrospray ionization mass spectrometry. Experiments were performed in biological duplicates.

    Techniques Used: Cell Culture, Fractionation, Labeling, Liquid Chromatography, Mass Spectrometry

    36) Product Images from "DamC reveals principles of chromatin folding in vivo without crosslinking and ligation"

    Article Title: DamC reveals principles of chromatin folding in vivo without crosslinking and ligation

    Journal: Nature structural & molecular biology

    doi: 10.1038/s41594-019-0231-0

    An inducible mESC line to perform DamC and test the model predictions. a. mESCs expressing rTetR-Dam-EGFP-ERT2 allow control of the nuclear concentration of the fusion protein by changing the amount of 4-hydroxy-tamoxifen (4-OHT) in the culture medium. b. Nuclear concentration of the rTetR-Dam fusion protein as a function of 4-OHT concentration in the polyclonal population with 890 insertions (blue) and in the subclone with 135 insertions (green). Number of protein copies per nucleus were determined using mass spectrometry on nuclear extracts and divided by the average nuclear volume (~490 fl) as determined using DAPI staining (see Supplementary Figure 2 ). Error bars are s.d. of two biological replicates (independent cell cultures). c. Random integration of large numbers of 50x TetO platforms using the piggyBac transposon. Accumulation of EGFP signal to nuclear foci in the presence of Dox (right: max. intensity projection over 10 Z planes) indicates binding of rTetR-Dam to the arrays and allows selecting clones with large numbers of insertions. d) Quantification of DamC experiments as a function of rTetR-Dam concentration in cells with 890 (upper panel, blue) and 135 (lower panel, green) TetO viewpoints. Blue data points, mean and s.d. from over the 100 TetO viewpoints with highest enrichment. Green data points, mean and s.d. from over 130 TetO viewpoints (5 viewpoints were excluded due to absence of DamC signal). Red line, model fit to the experimental data.
    Figure Legend Snippet: An inducible mESC line to perform DamC and test the model predictions. a. mESCs expressing rTetR-Dam-EGFP-ERT2 allow control of the nuclear concentration of the fusion protein by changing the amount of 4-hydroxy-tamoxifen (4-OHT) in the culture medium. b. Nuclear concentration of the rTetR-Dam fusion protein as a function of 4-OHT concentration in the polyclonal population with 890 insertions (blue) and in the subclone with 135 insertions (green). Number of protein copies per nucleus were determined using mass spectrometry on nuclear extracts and divided by the average nuclear volume (~490 fl) as determined using DAPI staining (see Supplementary Figure 2 ). Error bars are s.d. of two biological replicates (independent cell cultures). c. Random integration of large numbers of 50x TetO platforms using the piggyBac transposon. Accumulation of EGFP signal to nuclear foci in the presence of Dox (right: max. intensity projection over 10 Z planes) indicates binding of rTetR-Dam to the arrays and allows selecting clones with large numbers of insertions. d) Quantification of DamC experiments as a function of rTetR-Dam concentration in cells with 890 (upper panel, blue) and 135 (lower panel, green) TetO viewpoints. Blue data points, mean and s.d. from over the 100 TetO viewpoints with highest enrichment. Green data points, mean and s.d. from over 130 TetO viewpoints (5 viewpoints were excluded due to absence of DamC signal). Red line, model fit to the experimental data.

    Techniques Used: Expressing, Concentration Assay, Mass Spectrometry, Staining, Binding Assay, Clone Assay

    37) Product Images from "Aging Disrupts the Circadian Patterns of Protein Expression in the Murine Hippocampus"

    Article Title: Aging Disrupts the Circadian Patterns of Protein Expression in the Murine Hippocampus

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2019.00368

    Aging disrupts the hippocampal circadian proteome. (A) Experimental design and workflow of the MS-based analysis of proteins extracted from hippocampal tissues of young (9–10 weeks old) and middle-aged (44–52 weeks old) C57BL/6J mice. Samples were collected every 4 h over 2 days, and proteins extracted from tissues of individual mice were digested with trypsin, fractionated, and analyzed by an Orbitrap Elite mass spectrometer. (B) Proteome coverage: Venn diagram displaying the number of proteins quantified in at least two biological replicates per time point in young or middle-aged mice and overlap between ages. (C) Circadian proteins detected in young or middle-aged mice using the Perseus periodicity algorithm (period = 23.6 h; q -value
    Figure Legend Snippet: Aging disrupts the hippocampal circadian proteome. (A) Experimental design and workflow of the MS-based analysis of proteins extracted from hippocampal tissues of young (9–10 weeks old) and middle-aged (44–52 weeks old) C57BL/6J mice. Samples were collected every 4 h over 2 days, and proteins extracted from tissues of individual mice were digested with trypsin, fractionated, and analyzed by an Orbitrap Elite mass spectrometer. (B) Proteome coverage: Venn diagram displaying the number of proteins quantified in at least two biological replicates per time point in young or middle-aged mice and overlap between ages. (C) Circadian proteins detected in young or middle-aged mice using the Perseus periodicity algorithm (period = 23.6 h; q -value

    Techniques Used: Mouse Assay, Mass Spectrometry

    38) Product Images from "S-Glutathionylated Serine Proteinase Inhibitors as Plasma Biomarkers in Assessing Response to Redox-Modulating Drugs"

    Article Title: S-Glutathionylated Serine Proteinase Inhibitors as Plasma Biomarkers in Assessing Response to Redox-Modulating Drugs

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-11-4088

    S -glutathionylation of serpins A1 and A3 is time and dose dependent and impacts protein structure. Recombinant serpin A1 and A3 were treated with 1 mmol/L GSH and 40 µmol/L of PABA/NO for 0 to 30 minutes (A) or 0 to 100 µmol/L of PABA/NO
    Figure Legend Snippet: S -glutathionylation of serpins A1 and A3 is time and dose dependent and impacts protein structure. Recombinant serpin A1 and A3 were treated with 1 mmol/L GSH and 40 µmol/L of PABA/NO for 0 to 30 minutes (A) or 0 to 100 µmol/L of PABA/NO

    Techniques Used: Recombinant

    39) Product Images from "Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L"

    Article Title: Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L

    Journal: Genes & Development

    doi: 10.1101/gad.1977710

    Dok-7 Y396 and Y406 phosphopeptides bind to Crk-L and Crk in myotube lysates. ( A ) The amino acid sequences of the biotinylated Dok-7 phosphopeptides. ( B ) The Dok-7 biotin-pY406 phosphopeptide purified an ∼37-kDa doublet. The doublet failed to bind the nonphosphorylated biotin-Y406 peptide, and binding to the biotin-pY406 phosphopeptide was inhibited by addition of excess phosphopeptide. The ∼37-kDa doublet was identified as Crk-II and Crk-L by Q-TOF MS. ( C ) Western blots of myotube proteins isolated by binding to pY396 or pY406 phosphopeptides were probed with antibodies to Nck1/2, Crk-I, Crk-II, Crk-L, Abl, or Arg. Both phosphopeptides bound Crk-I, Crk-II, and Crk-L; in addition, the pY406 phosphopeptide bound Nck1/2. Neither phosphopeptide bound Abl or Arg. See also Supplemental Figure S2.
    Figure Legend Snippet: Dok-7 Y396 and Y406 phosphopeptides bind to Crk-L and Crk in myotube lysates. ( A ) The amino acid sequences of the biotinylated Dok-7 phosphopeptides. ( B ) The Dok-7 biotin-pY406 phosphopeptide purified an ∼37-kDa doublet. The doublet failed to bind the nonphosphorylated biotin-Y406 peptide, and binding to the biotin-pY406 phosphopeptide was inhibited by addition of excess phosphopeptide. The ∼37-kDa doublet was identified as Crk-II and Crk-L by Q-TOF MS. ( C ) Western blots of myotube proteins isolated by binding to pY396 or pY406 phosphopeptides were probed with antibodies to Nck1/2, Crk-I, Crk-II, Crk-L, Abl, or Arg. Both phosphopeptides bound Crk-I, Crk-II, and Crk-L; in addition, the pY406 phosphopeptide bound Nck1/2. Neither phosphopeptide bound Abl or Arg. See also Supplemental Figure S2.

    Techniques Used: Purification, Binding Assay, Mass Spectrometry, Western Blot, Isolation

    40) Product Images from "Demonstration of the Equivalent Pharmacokinetic/Pharmacodynamic Dosing Strategy in a Multiple-Dose Study of Gefitinib"

    Article Title: Demonstration of the Equivalent Pharmacokinetic/Pharmacodynamic Dosing Strategy in a Multiple-Dose Study of Gefitinib

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-09-0089

    Preclinical PK model of gefitinib in mice bearing wild-type and vIII mutant EGFR tumors
    Figure Legend Snippet: Preclinical PK model of gefitinib in mice bearing wild-type and vIII mutant EGFR tumors

    Techniques Used: Mouse Assay, Mutagenesis

    The PK model-predicted (―――) and mean (n = 3–4) observed (●) gefitinib concentrations for plasma (A), tumor (C) of LN229-wild-type EGFR tumor bearing group and plasma (B), tumor (D) of LN229-EGFRvIII mutant group,
    Figure Legend Snippet: The PK model-predicted (―――) and mean (n = 3–4) observed (●) gefitinib concentrations for plasma (A), tumor (C) of LN229-wild-type EGFR tumor bearing group and plasma (B), tumor (D) of LN229-EGFRvIII mutant group,

    Techniques Used: Mutagenesis

    Clinical PK/PD model simulations. Model simulations of gefitinib brain tumor concentration-time profiles (left y-axis) and corresponding tumor pERK inhibition-time profiles (right y-axis) following 1000 mg/d PO × 15 days in patients bearing intracerebral
    Figure Legend Snippet: Clinical PK/PD model simulations. Model simulations of gefitinib brain tumor concentration-time profiles (left y-axis) and corresponding tumor pERK inhibition-time profiles (right y-axis) following 1000 mg/d PO × 15 days in patients bearing intracerebral

    Techniques Used: Concentration Assay, Inhibition

    The PK/PD/tumor growth model-predicted (―――, gefitinib-treated group; ―――, vehicle control group) and mean observed (●, gefitinib-treated group, n = 23; ○, vehicle control group, n = 9) tumor
    Figure Legend Snippet: The PK/PD/tumor growth model-predicted (―――, gefitinib-treated group; ―――, vehicle control group) and mean observed (●, gefitinib-treated group, n = 23; ○, vehicle control group, n = 9) tumor

    Techniques Used:

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    Article Title: Protective Effect of a (Poly)phenol-Rich Extract Derived from Sweet Cherries Culls against Oxidative Cell Damage
    Article Snippet: .. HPLC-DAD-ED and LC-MS/MS Analysis HPLC-DAD-ED: The High Performance Liquid Chromatography (HPLC) system used was a Thermo Finnigan (Surveyor model) equipped with an autosampler, pump, diode-array detector (DAD) and electrochemical detector (ED) (Dionex, ED40). .. Chromatographic separation was carried out on a Lichrocart RP-18 column (250 × 4 mm, 5 µm, Merck) in a thermostated oven at 35 °C.

    Article Title: Use of Physiologically Based Biokinetic (PBBK) Modeling to Study Estragole Bioactivation and Detoxification in Humans as Compared with Male Rats
    Article Snippet: .. Formation of GS-1′-oxoestragole in the incubations was verified by LC-MS, which was performed on a Finnigan Surveyor HPLC system coupled to an LXQ mass spectrometer (Thermo Finnigan, San Jose, CA). .. Aliquots of 20 μl (injected volume) were separated on an Alltima C18 5-μm column, 150 x 2.1 mm (Alltech).

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Use of Physiologically Based Biokinetic (PBBK) Modeling to Study Estragole Bioactivation and Detoxification in Humans as Compared with Male Rats
    Article Snippet: .. Formation of GS-1′-oxoestragole in the incubations was verified by LC-MS, which was performed on a Finnigan Surveyor HPLC system coupled to an LXQ mass spectrometer (Thermo Finnigan, San Jose, CA). .. Aliquots of 20 μl (injected volume) were separated on an Alltima C18 5-μm column, 150 x 2.1 mm (Alltech).

    Isolation:

    Article Title: PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65
    Article Snippet: .. Isolated phosphopeptides were analysed by LC-MS-MS on a proxeon Easy-nLC nano liquid chromatography system coupled to a Thermo LTQ-orbitrap mass spectrometer. .. The resultant data files were searched using Mascot ( www.matrixscience.com ) run on an in-house system against a database containing the Parkin sequence, with a 10 ppm mass accuracy for precursor ions, a 0.8 Da tolerance for fragment ions, and allowing for Phospho (ST), Phospho (Y), Oxidation (M) and Dioxidation (M) as variable modifications.

    Construct:

    Article Title: Label-free quantitative phosphorylation analysis of human transgelin2 in Jurkat T cells reveals distinct phosphorylation patterns under PKA and PKC activation conditions
    Article Snippet: .. For the quantification of phosphorylation levels, selected ion chromatograms of identified phosphopeptides were constructed using Xcalibur 2.1.0 SP1 program (Thermo) and integrated peak areas were then calculated with a built-in feature of Xcalibur program for comparison purposes. .. Relative quantification of each phosphopeptide was performed by comparing peak-areas of no-activation condition and those of two kinase activation condition samples.

    Mass Spectrometry:

    Article Title: Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1
    Article Snippet: .. Mass spectrometry LC‐MS/MS analysis was performed on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). .. Peptide separation was carried out by a Proxeon EASY‐nLC II liquid chromatography system (Thermo Fisher Scientific) connected to an RP‐HPLC column (75 μm x 10 cm) packed with Magic C18 AQ (3 μm) resin (WICOM International, Maienfeld, Switzerland).

    Article Title: A proteomic profiling dataset of recombinant Chinese hamster ovary cells showing enhanced cellular growth following miR-378 depletion
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    Article Title: PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65
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    Article Title: Use of Physiologically Based Biokinetic (PBBK) Modeling to Study Estragole Bioactivation and Detoxification in Humans as Compared with Male Rats
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    Sequencing:

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    Liquid Chromatography:

    Article Title: A proteomic profiling dataset of recombinant Chinese hamster ovary cells showing enhanced cellular growth following miR-378 depletion
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    Article Title: PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65
    Article Snippet: .. Isolated phosphopeptides were analysed by LC-MS-MS on a proxeon Easy-nLC nano liquid chromatography system coupled to a Thermo LTQ-orbitrap mass spectrometer. .. The resultant data files were searched using Mascot ( www.matrixscience.com ) run on an in-house system against a database containing the Parkin sequence, with a 10 ppm mass accuracy for precursor ions, a 0.8 Da tolerance for fragment ions, and allowing for Phospho (ST), Phospho (Y), Oxidation (M) and Dioxidation (M) as variable modifications.

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    Thermo Fisher lc ms ms analysis all peptide
    GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif <t>analysis</t> for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr <t>peptide</t> abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) <t>MS/MS</t> spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).
    Lc Ms Ms Analysis All Peptide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher lc ms analysis lc ms
    Chromatographic profile of the Impatiens glandulifera Royle methanolic extract of the roots measured as total ion current (TIC) by <t>LC-MS</t> (APCI). MS spectrum of THNG is shown on the Figure 3 .
    Lc Ms Analysis Lc Ms, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher lc esi ms ms
    Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB <t>p65</t> antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by <t>LC-ESI-MS/MS.</t> The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.
    Lc Esi Ms Ms, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mass spectrometry lc ms
    A systematic affinity purification <t>mass</t> <t>spectrometry</t> ( AP ‐ <t>MS</t> ) approach to define the human Hpo pathway interaction proteome Selection of primary and secondary baits in this study. Baits were selected sequentially, starting with the core components of the Hpo kinase signaling pathway and extended based on obtained AP ‐ MS results or homology to D rosophila H po components. Biochemical workflow for native protein complex purification from HEK 293‐Flp T‐rex cells. Bait proteins were expressed from a tetracycline‐inducible CMV promoter, with a N‐terminal Strep‐ HA fusion tag following induction with doxycycline for 24 h. Cells were lysed, complexes affinity‐purified and processed for analysis by tandem mass spectrometry. Data analysis pipeline. Acquired mass spectra from 90 experiments (at least 2 biological replicates per bait) were searched with X!Tandem. Search results were statistically validated by the Trans‐Proteomic Pipeline ( TPP ) to match a protein identification false discovery rate of
    Mass Spectrometry Lc Ms, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).

    Journal: Journal of Proteome Research

    Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

    doi: 10.1021/acs.jproteome.8b00895

    Figure Lengend Snippet: GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).

    Article Snippet: LC–MS/MS Analysis All peptide samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer fronted with an EASY-Spray Source, coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific).

    Techniques: Sequencing, Mass Spectrometry

    Data processing of product ion triggered MS/MS spectra. (A) A schematic of SEQUEST-HT searches of triggered EThcD and HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Number of peptide-spectrum matches (PSMs) of assigned ADPr and unmodified peptides from the triggered spectra. (C–E) Distribution of isolation interference for product ion triggered or DDA PSMs. (F) Number of ADPr peptides with high confidence detected by either EThcD or HCD. (G) Venn diagrams comparing ADPr peptide identifications between EThcD and HCD for all ADPr peptides, and those with > 95% ADPr acceptor site probability.

    Journal: Journal of Proteome Research

    Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

    doi: 10.1021/acs.jproteome.8b00895

    Figure Lengend Snippet: Data processing of product ion triggered MS/MS spectra. (A) A schematic of SEQUEST-HT searches of triggered EThcD and HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Number of peptide-spectrum matches (PSMs) of assigned ADPr and unmodified peptides from the triggered spectra. (C–E) Distribution of isolation interference for product ion triggered or DDA PSMs. (F) Number of ADPr peptides with high confidence detected by either EThcD or HCD. (G) Venn diagrams comparing ADPr peptide identifications between EThcD and HCD for all ADPr peptides, and those with > 95% ADPr acceptor site probability.

    Article Snippet: LC–MS/MS Analysis All peptide samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer fronted with an EASY-Spray Source, coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific).

    Techniques: Mass Spectrometry, Isolation

    Chromatographic profile of the Impatiens glandulifera Royle methanolic extract of the roots measured as total ion current (TIC) by LC-MS (APCI). MS spectrum of THNG is shown on the Figure 3 .

    Journal: Molecules

    Article Title: Separation and Identification of 1,2,4-Trihydroxynaphthalene-1-O-glucoside in Impatiens glandulifera Royle

    doi: 10.3390/molecules18078429

    Figure Lengend Snippet: Chromatographic profile of the Impatiens glandulifera Royle methanolic extract of the roots measured as total ion current (TIC) by LC-MS (APCI). MS spectrum of THNG is shown on the Figure 3 .

    Article Snippet: LC-MS Analysis LC-MS was performed using LCQ Accela Fleet (Thermo Fisher Scientific, San Jose, CA, USA) with atmospheric pressure chemical (APCI) and a photodiode array detector.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB p65 antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by LC-ESI-MS/MS. The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.

    Journal: PLoS ONE

    Article Title: Hsc70 Is a Novel Interactor of NF-kappaB p65 in Living Hippocampal Neurons

    doi: 10.1371/journal.pone.0065280

    Figure Lengend Snippet: Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB p65 antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by LC-ESI-MS/MS. The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.

    Article Snippet: MALDI-MS and LC-ESI-MS/MS revealed an interaction of p65 with compounds of the endocytosis network: clathrin and dynamin-1, microtubule subunits or associated proteins like beta 5-tubulin, tubulin alpha 6, beta actin, dihydropyrimidinase-related protein 2, neurofilament and light polypeptide (NEFL) and heat shock proteins HSP90 alpha class A and B (data not shown).

    Techniques: Immunoprecipitation, SDS-Gel, Mass Spectrometry, Transfection, Western Blot, Cotransfection

    A systematic affinity purification mass spectrometry ( AP ‐ MS ) approach to define the human Hpo pathway interaction proteome Selection of primary and secondary baits in this study. Baits were selected sequentially, starting with the core components of the Hpo kinase signaling pathway and extended based on obtained AP ‐ MS results or homology to D rosophila H po components. Biochemical workflow for native protein complex purification from HEK 293‐Flp T‐rex cells. Bait proteins were expressed from a tetracycline‐inducible CMV promoter, with a N‐terminal Strep‐ HA fusion tag following induction with doxycycline for 24 h. Cells were lysed, complexes affinity‐purified and processed for analysis by tandem mass spectrometry. Data analysis pipeline. Acquired mass spectra from 90 experiments (at least 2 biological replicates per bait) were searched with X!Tandem. Search results were statistically validated by the Trans‐Proteomic Pipeline ( TPP ) to match a protein identification false discovery rate of

    Journal: Molecular Systems Biology

    Article Title: Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1

    doi: 10.1002/msb.201304750

    Figure Lengend Snippet: A systematic affinity purification mass spectrometry ( AP ‐ MS ) approach to define the human Hpo pathway interaction proteome Selection of primary and secondary baits in this study. Baits were selected sequentially, starting with the core components of the Hpo kinase signaling pathway and extended based on obtained AP ‐ MS results or homology to D rosophila H po components. Biochemical workflow for native protein complex purification from HEK 293‐Flp T‐rex cells. Bait proteins were expressed from a tetracycline‐inducible CMV promoter, with a N‐terminal Strep‐ HA fusion tag following induction with doxycycline for 24 h. Cells were lysed, complexes affinity‐purified and processed for analysis by tandem mass spectrometry. Data analysis pipeline. Acquired mass spectra from 90 experiments (at least 2 biological replicates per bait) were searched with X!Tandem. Search results were statistically validated by the Trans‐Proteomic Pipeline ( TPP ) to match a protein identification false discovery rate of

    Article Snippet: Mass spectrometry LC‐MS/MS analysis was performed on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific).

    Techniques: Affinity Purification, Mass Spectrometry, Selection, Purification