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  • 99
    Thermo Fisher dithiothreito
    Characterization of Atg4B activity using synthetic peptide substrates. (A) Various concentrations of recombinant Atg4B were incubated with 100 µM synthetic tetrapeptides conjugated with AFC in 50 mM <t>Tris-HCl,</t> pH 8.0, 5 mM <t>DTT</t> at 37°C for
    Dithiothreito, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dithiothreitol sputolysin
    Characterization of Atg4B activity using synthetic peptide substrates. (A) Various concentrations of recombinant Atg4B were incubated with 100 µM synthetic tetrapeptides conjugated with AFC in 50 mM <t>Tris-HCl,</t> pH 8.0, 5 mM <t>DTT</t> at 37°C for
    Dithiothreitol Sputolysin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA dithiothreitol
    In vitro release behavior of PTX from MPEG-SS-2SA/TPGS, MPEG-2SA/TPGS, and MPEG-SS-2SA micelles in PBS solution with ( A ) or without ( B ) 10 mm DTT. Abbreviations: DTT, <t>dithiothreitol;</t> MPEG, poly (ethylene glycol) monomethyl ether; PBS, phosphate-buffered saline; PTX, paclitaxel; TEM, transmission electron microscopy; TPGS, d -α-tocopheryl polyethylene glycol succinate; SA, stearic acid; h, hours.
    Dithiothreitol, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Boehringer Ingelheim sputolysin
    In vitro release behavior of PTX from MPEG-SS-2SA/TPGS, MPEG-2SA/TPGS, and MPEG-SS-2SA micelles in PBS solution with ( A ) or without ( B ) 10 mm DTT. Abbreviations: DTT, <t>dithiothreitol;</t> MPEG, poly (ethylene glycol) monomethyl ether; PBS, phosphate-buffered saline; PTX, paclitaxel; TEM, transmission electron microscopy; TPGS, d -α-tocopheryl polyethylene glycol succinate; SA, stearic acid; h, hours.
    Sputolysin, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck & Co dithiothreitol
    Role of disulfide bridges in SNMP1 trafficking and function. ( a ) Analysis of the presence of intramolecular disulfide bonds in SNMP1 by electrophoretic mobility assay in non-reducing versus reducing conditions. Total antennal protein extracts were resolved by 4–20% SDS–PAGE in the presence or absence of the reducing agents <t>dithiothreitol</t> (100 mM) or β-mercaptoethanol (10%). After western blotting, immunodetection was performed with α-SNMP1. ( b ) Analysis of the rescue properties of SNMP1 mutants lacking one or more disulfide-bond forming cysteine residues as indicated in the cartoons. Left: immunostaining with α-SNMP1 on antennal cryosections. Scale bars, 20 μm. Centre: representative traces of electrophysiological recordings of OR67d neurons in male flies stimulated with 10% cVA. Right: mean neuronal responses±s.e.m. in each genotype. There are significant statistical differences in neuronal responses due to genotype for both 1 and 10% cVA (Kruskal–Wallis, P
    Dithiothreitol, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Avantor dithiothreitol
    Role of disulfide bridges in SNMP1 trafficking and function. ( a ) Analysis of the presence of intramolecular disulfide bonds in SNMP1 by electrophoretic mobility assay in non-reducing versus reducing conditions. Total antennal protein extracts were resolved by 4–20% SDS–PAGE in the presence or absence of the reducing agents <t>dithiothreitol</t> (100 mM) or β-mercaptoethanol (10%). After western blotting, immunodetection was performed with α-SNMP1. ( b ) Analysis of the rescue properties of SNMP1 mutants lacking one or more disulfide-bond forming cysteine residues as indicated in the cartoons. Left: immunostaining with α-SNMP1 on antennal cryosections. Scale bars, 20 μm. Centre: representative traces of electrophysiological recordings of OR67d neurons in male flies stimulated with 10% cVA. Right: mean neuronal responses±s.e.m. in each genotype. There are significant statistical differences in neuronal responses due to genotype for both 1 and 10% cVA (Kruskal–Wallis, P
    Dithiothreitol, supplied by Avantor, used in various techniques. Bioz Stars score: 92/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Behring Diagnostics sputolysin
    Role of disulfide bridges in SNMP1 trafficking and function. ( a ) Analysis of the presence of intramolecular disulfide bonds in SNMP1 by electrophoretic mobility assay in non-reducing versus reducing conditions. Total antennal protein extracts were resolved by 4–20% SDS–PAGE in the presence or absence of the reducing agents <t>dithiothreitol</t> (100 mM) or β-mercaptoethanol (10%). After western blotting, immunodetection was performed with α-SNMP1. ( b ) Analysis of the rescue properties of SNMP1 mutants lacking one or more disulfide-bond forming cysteine residues as indicated in the cartoons. Left: immunostaining with α-SNMP1 on antennal cryosections. Scale bars, 20 μm. Centre: representative traces of electrophysiological recordings of OR67d neurons in male flies stimulated with 10% cVA. Right: mean neuronal responses±s.e.m. in each genotype. There are significant statistical differences in neuronal responses due to genotype for both 1 and 10% cVA (Kruskal–Wallis, P
    Sputolysin, supplied by Behring Diagnostics, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad dithiothreito
    Western immunoblot demonstrating the recognition of recombinant PgPM4 and plasmepsin 4 (PgPM4) in P. gallinaceum ookinete conditioned medium and lysate by mAb 1H10. −, unreduced condition; +, reducing conditions with <t>dithiothreitol.</t>
    Dithiothreito, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dithiothreito/product/Bio-Rad
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    99
    Millipore dithiothreitol
    Relationship between RESET and the UPR, Related to Figure 7 (A) Extended data set for Figure 7 A. NRK cells stably expressing the XBP1-mCherry UPR reporter and GFP-PrP ∗ were treated with 0.5 mM <t>dithiothreitol</t> (DTT). Time series were collected over the course of 18 hr starting 5 min after addition of DTT. For each time point, both GFP-PrP ∗ and XBP1-mCh were imaged. Scale bar, 10 μm. (B) Flp-In T-REx HEK293 cells (Invitrogen) expressing wild-type human PrP at the FRT locus were induced with the indicated concentrations of doxycycline for 24 hr. Total cell lysates were analyzed by immunoblotting with the 3F4 monoclonal antibody, using total hamster brain homogenate as a standard. Loading was controlled by β-tubulin immunoblot. Similar levels of expression of PrP were seen with transient transfection and doxycycline induction in the Flp-In T-REx HEK293 cells. (C) Flp-In T-REx HEK293 cells, either untransfected, or stably transfected with the indicated PrP construct, were transfected with an ATF6-luciferase UPR reporter (or control plasmid), then induced with 5 ng/ml doxycycline for 16 hr. Cells were then assessed for luciferase activity. Five individual wells of cells were measured for each condition (gray bars). The mean of the five replicates (±SD) is shown with the black bars, and indicated below the graph. (D) Flp-In T-REx HEK293 cells, which were either untransfected (UT) or stably transfected with the indicated inducible PrP construct (C179A is YFP-PrP ∗ ), were transfected with an ATF6-luciferase reporter. Expression of PrP constructs was induced with 5 ng/ml doxycycline for 16 hr. Triplicate wells were then assessed for luciferase activity (mean ± SD). In parallel, Flp-In T-REx HEK293 cells were transfected with an ATF6-luciferase reporter, treated with the indicated concentrations of dithiothreitol (DTT) for 16 hr, and analyzed in triplicate for luciferase activity (mean ± SD).
    Dithiothreitol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sputolysin reagent
    Positive correlation of BPI and IL-8 concentrations in cell-free lung fluid from CF patients. Supernatants of sputum samples from CF patients were collected after treatment with <t>sputolysin</t> and subsequent centrifugation. IL-8 and BPI levels were determined by ELISA. Linear regression shows the positive correlation r 2 = 0.6785. The P value was
    Sputolysin Reagent, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Caldon Biotech Inc sputolysin
    Positive correlation of BPI and IL-8 concentrations in cell-free lung fluid from CF patients. Supernatants of sputum samples from CF patients were collected after treatment with <t>sputolysin</t> and subsequent centrifugation. IL-8 and BPI levels were determined by ELISA. Linear regression shows the positive correlation r 2 = 0.6785. The P value was
    Sputolysin, supplied by Caldon Biotech Inc, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Dade Behring sputolysin
    Positive correlation of BPI and IL-8 concentrations in cell-free lung fluid from CF patients. Supernatants of sputum samples from CF patients were collected after treatment with <t>sputolysin</t> and subsequent centrifugation. IL-8 and BPI levels were determined by ELISA. Linear regression shows the positive correlation r 2 = 0.6785. The P value was
    Sputolysin, supplied by Dade Behring, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sputolysin solution
    Positive correlation of BPI and IL-8 concentrations in cell-free lung fluid from CF patients. Supernatants of sputum samples from CF patients were collected after treatment with <t>sputolysin</t> and subsequent centrifugation. IL-8 and BPI levels were determined by ELISA. Linear regression shows the positive correlation r 2 = 0.6785. The P value was
    Sputolysin Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Characterization of Atg4B activity using synthetic peptide substrates. (A) Various concentrations of recombinant Atg4B were incubated with 100 µM synthetic tetrapeptides conjugated with AFC in 50 mM Tris-HCl, pH 8.0, 5 mM DTT at 37°C for

    Journal: Autophagy

    Article Title: Synthetic substrates for measuring activity of autophagy proteases

    doi: 10.4161/auto.6.7.13075

    Figure Lengend Snippet: Characterization of Atg4B activity using synthetic peptide substrates. (A) Various concentrations of recombinant Atg4B were incubated with 100 µM synthetic tetrapeptides conjugated with AFC in 50 mM Tris-HCl, pH 8.0, 5 mM DTT at 37°C for

    Article Snippet: Briefly, recombinant Atg4B was mixed with 100 nM LC3B-PLA2 fusion protein in 20 µl PLA2 reaction buffer containing 20 mM Tris-HCl, pH 8.0, 2 mM CaCl and 1 mM DTT and 20 µM 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino) hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-C6 -HPC) (Invitrogen, N-3786).

    Techniques: Activity Assay, Recombinant, Incubation

    In vitro release behavior of PTX from MPEG-SS-2SA/TPGS, MPEG-2SA/TPGS, and MPEG-SS-2SA micelles in PBS solution with ( A ) or without ( B ) 10 mm DTT. Abbreviations: DTT, dithiothreitol; MPEG, poly (ethylene glycol) monomethyl ether; PBS, phosphate-buffered saline; PTX, paclitaxel; TEM, transmission electron microscopy; TPGS, d -α-tocopheryl polyethylene glycol succinate; SA, stearic acid; h, hours.

    Journal: International Journal of Nanomedicine

    Article Title: Biodegradable mixed MPEG-SS-2SA/TPGS micelles for triggered intracellular release of paclitaxel and reversing multidrug resistance

    doi: 10.2147/IJN.S111930

    Figure Lengend Snippet: In vitro release behavior of PTX from MPEG-SS-2SA/TPGS, MPEG-2SA/TPGS, and MPEG-SS-2SA micelles in PBS solution with ( A ) or without ( B ) 10 mm DTT. Abbreviations: DTT, dithiothreitol; MPEG, poly (ethylene glycol) monomethyl ether; PBS, phosphate-buffered saline; PTX, paclitaxel; TEM, transmission electron microscopy; TPGS, d -α-tocopheryl polyethylene glycol succinate; SA, stearic acid; h, hours.

    Article Snippet: Dithiothreitol (DTT, 99%) was purchased from Merck (Darmstadt, Germany).

    Techniques: In Vitro, Transmission Electron Microscopy, Transmission Assay, Electron Microscopy

    Role of disulfide bridges in SNMP1 trafficking and function. ( a ) Analysis of the presence of intramolecular disulfide bonds in SNMP1 by electrophoretic mobility assay in non-reducing versus reducing conditions. Total antennal protein extracts were resolved by 4–20% SDS–PAGE in the presence or absence of the reducing agents dithiothreitol (100 mM) or β-mercaptoethanol (10%). After western blotting, immunodetection was performed with α-SNMP1. ( b ) Analysis of the rescue properties of SNMP1 mutants lacking one or more disulfide-bond forming cysteine residues as indicated in the cartoons. Left: immunostaining with α-SNMP1 on antennal cryosections. Scale bars, 20 μm. Centre: representative traces of electrophysiological recordings of OR67d neurons in male flies stimulated with 10% cVA. Right: mean neuronal responses±s.e.m. in each genotype. There are significant statistical differences in neuronal responses due to genotype for both 1 and 10% cVA (Kruskal–Wallis, P

    Journal: Nature Communications

    Article Title: A CD36 ectodomain mediates insect pheromone detection via a putative tunnelling mechanism

    doi: 10.1038/ncomms11866

    Figure Lengend Snippet: Role of disulfide bridges in SNMP1 trafficking and function. ( a ) Analysis of the presence of intramolecular disulfide bonds in SNMP1 by electrophoretic mobility assay in non-reducing versus reducing conditions. Total antennal protein extracts were resolved by 4–20% SDS–PAGE in the presence or absence of the reducing agents dithiothreitol (100 mM) or β-mercaptoethanol (10%). After western blotting, immunodetection was performed with α-SNMP1. ( b ) Analysis of the rescue properties of SNMP1 mutants lacking one or more disulfide-bond forming cysteine residues as indicated in the cartoons. Left: immunostaining with α-SNMP1 on antennal cryosections. Scale bars, 20 μm. Centre: representative traces of electrophysiological recordings of OR67d neurons in male flies stimulated with 10% cVA. Right: mean neuronal responses±s.e.m. in each genotype. There are significant statistical differences in neuronal responses due to genotype for both 1 and 10% cVA (Kruskal–Wallis, P

    Article Snippet: To analyse the presence of disulfide bonds in SNMP1, protein extracts were incubated in the presence or absence of either 100 mM dithiothreitol (Merck) or 10% β-mercaptoethanol (Promega) during 3 min at 95 °C.

    Techniques: SDS Page, Western Blot, Immunodetection, Immunostaining

    Western immunoblot demonstrating the recognition of recombinant PgPM4 and plasmepsin 4 (PgPM4) in P. gallinaceum ookinete conditioned medium and lysate by mAb 1H10. −, unreduced condition; +, reducing conditions with dithiothreitol.

    Journal: The Journal of Biological Chemistry

    Article Title: Apical Surface Expression of Aspartic Protease Plasmepsin 4, a Potential Transmission-blocking Target of the Plasmodium Ookinete *

    doi: 10.1074/jbc.M109.063388

    Figure Lengend Snippet: Western immunoblot demonstrating the recognition of recombinant PgPM4 and plasmepsin 4 (PgPM4) in P. gallinaceum ookinete conditioned medium and lysate by mAb 1H10. −, unreduced condition; +, reducing conditions with dithiothreitol.

    Article Snippet: Resolved proteins were equilibrated for 15 min in buffer I consisting of 6 m urea, 0.376 m Tris-HCl, pH 9.4, 2% SDS, 2% dithiothreitol and buffer II consisting of 6 m urea, 0.376 m Tris-HCl, pH 9.4, 2% SDS, 3% iodoacetamide and then washed with SDS running buffer and separated with SDS-PAGE using 12.5% Tris-HCl gels in a Protean II apparatus (Bio-Rad) operated at 200 V for 57 min. Mass spectrometry-compatible silver staining (Silverquest silver staining kit, Invitrogen) was used to detect proteins.

    Techniques: Western Blot, Recombinant

    LC-MS/MS analysis of the tryptic peptide containing Cys60 of LeACO1 and LeACO2 recombinant protein (A) . The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum are highlighted in red. A 32 D mass shift in Cys60 were included in the analysis of data by Maxquant software. Immunoblot analysis of recombinant LeACO1 mutation proteins (B) . NaHS-induced persulfidation of LeACO1 mutation was detected using a MBST. Recombinant proteins were pretreated with NaHS (200 μM) and NaHS plus DTT (10 mM) for 20 min, and then the immunoblot analysis carried out. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Input is anti-his signal, which indicate the total protein amount. Analysis of recombinant LeACO1 C60S mutation protein activity (C) . Recombinant proteins were pretreated with NaHS and NaHS plus 10 mM DTT for 20 min, and then enzyme activity was analyzed. Mean values ± SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P

    Journal: Frontiers in Plant Science

    Article Title: Ethylene-Induced Hydrogen Sulfide Negatively Regulates Ethylene Biosynthesis by Persulfidation of ACO in Tomato Under Osmotic Stress

    doi: 10.3389/fpls.2018.01517

    Figure Lengend Snippet: LC-MS/MS analysis of the tryptic peptide containing Cys60 of LeACO1 and LeACO2 recombinant protein (A) . The table inside the spectrum contains the predicted ion types for the modified peptide, and the ions detected in the spectrum are highlighted in red. A 32 D mass shift in Cys60 were included in the analysis of data by Maxquant software. Immunoblot analysis of recombinant LeACO1 mutation proteins (B) . NaHS-induced persulfidation of LeACO1 mutation was detected using a MBST. Recombinant proteins were pretreated with NaHS (200 μM) and NaHS plus DTT (10 mM) for 20 min, and then the immunoblot analysis carried out. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Input is anti-his signal, which indicate the total protein amount. Analysis of recombinant LeACO1 C60S mutation protein activity (C) . Recombinant proteins were pretreated with NaHS and NaHS plus 10 mM DTT for 20 min, and then enzyme activity was analyzed. Mean values ± SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P

    Article Snippet: The purified recombinant LeACO1 and LeACO2 proteins were treated with 50 to 400 μM NaHS to increase the concentration of persulfidated protein or with 10 mM DTT to reduce all of the disulfide bonds; both treatments were carried out at 4°C for 20 min. NaHS was removed using Micro BioSpinP6columns (BioRad).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Recombinant, Modification, Software, Mutagenesis, Labeling, Activity Assay

    Immunoblot and activity analysis of recombinant LeACO1 and LeACO2 proteins. (A) NaHS-induced persulfidation of LeACO1 was detected using a MBST. (B) Quantification of persulfidation levels that are shown in (A) . (C) NaHS-induced persulfidation of LeACO2 was detected using a MBST. (D) Quantification of persulfidation levels that are shown in (C) . Input is anti-his signal, which indicate the total protein amount. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Total proteins were analyzed using an anti-his antibody. Effect of NaHS-induced persulfidation on LeACO1 (E) and LeACO2 (F) activity. Recombinant proteins were pretreated with NaHS and NaHS plus DTT (10 mM) for 20 min, and then the immunoblot analysis carried out. Mean values ± SE are calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P

    Journal: Frontiers in Plant Science

    Article Title: Ethylene-Induced Hydrogen Sulfide Negatively Regulates Ethylene Biosynthesis by Persulfidation of ACO in Tomato Under Osmotic Stress

    doi: 10.3389/fpls.2018.01517

    Figure Lengend Snippet: Immunoblot and activity analysis of recombinant LeACO1 and LeACO2 proteins. (A) NaHS-induced persulfidation of LeACO1 was detected using a MBST. (B) Quantification of persulfidation levels that are shown in (A) . (C) NaHS-induced persulfidation of LeACO2 was detected using a MBST. (D) Quantification of persulfidation levels that are shown in (C) . Input is anti-his signal, which indicate the total protein amount. Persulfidated proteins were labeled with biotin and analyzed using an anti-biotin antibody. Total proteins were analyzed using an anti-his antibody. Effect of NaHS-induced persulfidation on LeACO1 (E) and LeACO2 (F) activity. Recombinant proteins were pretreated with NaHS and NaHS plus DTT (10 mM) for 20 min, and then the immunoblot analysis carried out. Mean values ± SE are calculated from three replicates. Within each set of experiments, bars with different letters are significant different (Duncan’s multiple range tests, P

    Article Snippet: The purified recombinant LeACO1 and LeACO2 proteins were treated with 50 to 400 μM NaHS to increase the concentration of persulfidated protein or with 10 mM DTT to reduce all of the disulfide bonds; both treatments were carried out at 4°C for 20 min. NaHS was removed using Micro BioSpinP6columns (BioRad).

    Techniques: Activity Assay, Recombinant, Labeling

    Relationship between RESET and the UPR, Related to Figure 7 (A) Extended data set for Figure 7 A. NRK cells stably expressing the XBP1-mCherry UPR reporter and GFP-PrP ∗ were treated with 0.5 mM dithiothreitol (DTT). Time series were collected over the course of 18 hr starting 5 min after addition of DTT. For each time point, both GFP-PrP ∗ and XBP1-mCh were imaged. Scale bar, 10 μm. (B) Flp-In T-REx HEK293 cells (Invitrogen) expressing wild-type human PrP at the FRT locus were induced with the indicated concentrations of doxycycline for 24 hr. Total cell lysates were analyzed by immunoblotting with the 3F4 monoclonal antibody, using total hamster brain homogenate as a standard. Loading was controlled by β-tubulin immunoblot. Similar levels of expression of PrP were seen with transient transfection and doxycycline induction in the Flp-In T-REx HEK293 cells. (C) Flp-In T-REx HEK293 cells, either untransfected, or stably transfected with the indicated PrP construct, were transfected with an ATF6-luciferase UPR reporter (or control plasmid), then induced with 5 ng/ml doxycycline for 16 hr. Cells were then assessed for luciferase activity. Five individual wells of cells were measured for each condition (gray bars). The mean of the five replicates (±SD) is shown with the black bars, and indicated below the graph. (D) Flp-In T-REx HEK293 cells, which were either untransfected (UT) or stably transfected with the indicated inducible PrP construct (C179A is YFP-PrP ∗ ), were transfected with an ATF6-luciferase reporter. Expression of PrP constructs was induced with 5 ng/ml doxycycline for 16 hr. Triplicate wells were then assessed for luciferase activity (mean ± SD). In parallel, Flp-In T-REx HEK293 cells were transfected with an ATF6-luciferase reporter, treated with the indicated concentrations of dithiothreitol (DTT) for 16 hr, and analyzed in triplicate for luciferase activity (mean ± SD).

    Journal: Cell

    Article Title: ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway

    doi: 10.1016/j.cell.2014.06.026

    Figure Lengend Snippet: Relationship between RESET and the UPR, Related to Figure 7 (A) Extended data set for Figure 7 A. NRK cells stably expressing the XBP1-mCherry UPR reporter and GFP-PrP ∗ were treated with 0.5 mM dithiothreitol (DTT). Time series were collected over the course of 18 hr starting 5 min after addition of DTT. For each time point, both GFP-PrP ∗ and XBP1-mCh were imaged. Scale bar, 10 μm. (B) Flp-In T-REx HEK293 cells (Invitrogen) expressing wild-type human PrP at the FRT locus were induced with the indicated concentrations of doxycycline for 24 hr. Total cell lysates were analyzed by immunoblotting with the 3F4 monoclonal antibody, using total hamster brain homogenate as a standard. Loading was controlled by β-tubulin immunoblot. Similar levels of expression of PrP were seen with transient transfection and doxycycline induction in the Flp-In T-REx HEK293 cells. (C) Flp-In T-REx HEK293 cells, either untransfected, or stably transfected with the indicated PrP construct, were transfected with an ATF6-luciferase UPR reporter (or control plasmid), then induced with 5 ng/ml doxycycline for 16 hr. Cells were then assessed for luciferase activity. Five individual wells of cells were measured for each condition (gray bars). The mean of the five replicates (±SD) is shown with the black bars, and indicated below the graph. (D) Flp-In T-REx HEK293 cells, which were either untransfected (UT) or stably transfected with the indicated inducible PrP construct (C179A is YFP-PrP ∗ ), were transfected with an ATF6-luciferase reporter. Expression of PrP constructs was induced with 5 ng/ml doxycycline for 16 hr. Triplicate wells were then assessed for luciferase activity (mean ± SD). In parallel, Flp-In T-REx HEK293 cells were transfected with an ATF6-luciferase reporter, treated with the indicated concentrations of dithiothreitol (DTT) for 16 hr, and analyzed in triplicate for luciferase activity (mean ± SD).

    Article Snippet: Drug Treatments Working concentrations are 0.1 μM thapsigargin, 0.5 mM dithiothreitol, 5 mM methyl-β-cyclodextrin, and 1 μM brefeldin A. Lysosomal protease inhibitors refer to 125 μM leupeptin + protease inhibitor cocktail (Sigma P8340) diluted 1:100.

    Techniques: Stable Transfection, Expressing, Transfection, Construct, Luciferase, Plasmid Preparation, Activity Assay

    ER Stress Induces Rapid Relocalization and Degradation of ER-Retained Misfolded PrP (A) Diagrams of YFP-tagged wild-type PrP (YFP-PrP) and YFP-tagged misfolded PrP (YFP-PrP ∗ ) depicting the GPI-anchor (yellow), two N-linked glycans (blue), the disulfide bond (black), and the YFP-tag (green). YFP-PrP ∗ lacks the intramolecular disulfide bond. (B) Steady-state localization of YFP-PrP (left) and YFP-PrP ∗ (right). CFP-KDEL marks the ER. (C) Time-lapse images of YFP-PrP ∗ -expressing cells after treatment with thapsigargin (TG, top) or with dithiothreitol (DTT, bottom) (D) Steady-state chase experiment performed using YFP-PrP ∗ -expressing cells. The top panel shows an autoradiograph of YFP-PrP ∗ immunoprecipitations from a representative experiment, and the bottom panel shows quantification from multiple experiments (mean ± SE; n = 4). Scale bars, 10 μm. See also Movies S1 and S2 and Figure S1 .

    Journal: Cell

    Article Title: ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway

    doi: 10.1016/j.cell.2014.06.026

    Figure Lengend Snippet: ER Stress Induces Rapid Relocalization and Degradation of ER-Retained Misfolded PrP (A) Diagrams of YFP-tagged wild-type PrP (YFP-PrP) and YFP-tagged misfolded PrP (YFP-PrP ∗ ) depicting the GPI-anchor (yellow), two N-linked glycans (blue), the disulfide bond (black), and the YFP-tag (green). YFP-PrP ∗ lacks the intramolecular disulfide bond. (B) Steady-state localization of YFP-PrP (left) and YFP-PrP ∗ (right). CFP-KDEL marks the ER. (C) Time-lapse images of YFP-PrP ∗ -expressing cells after treatment with thapsigargin (TG, top) or with dithiothreitol (DTT, bottom) (D) Steady-state chase experiment performed using YFP-PrP ∗ -expressing cells. The top panel shows an autoradiograph of YFP-PrP ∗ immunoprecipitations from a representative experiment, and the bottom panel shows quantification from multiple experiments (mean ± SE; n = 4). Scale bars, 10 μm. See also Movies S1 and S2 and Figure S1 .

    Article Snippet: Drug Treatments Working concentrations are 0.1 μM thapsigargin, 0.5 mM dithiothreitol, 5 mM methyl-β-cyclodextrin, and 1 μM brefeldin A. Lysosomal protease inhibitors refer to 125 μM leupeptin + protease inhibitor cocktail (Sigma P8340) diluted 1:100.

    Techniques: Expressing, Autoradiography

    Positive correlation of BPI and IL-8 concentrations in cell-free lung fluid from CF patients. Supernatants of sputum samples from CF patients were collected after treatment with sputolysin and subsequent centrifugation. IL-8 and BPI levels were determined by ELISA. Linear regression shows the positive correlation r 2 = 0.6785. The P value was

    Journal: Infection and Immunity

    Article Title: Expression and Antimicrobial Function of Bactericidal Permeability-Increasing Protein in Cystic Fibrosis Patients

    doi: 10.1128/IAI.02066-05

    Figure Lengend Snippet: Positive correlation of BPI and IL-8 concentrations in cell-free lung fluid from CF patients. Supernatants of sputum samples from CF patients were collected after treatment with sputolysin and subsequent centrifugation. IL-8 and BPI levels were determined by ELISA. Linear regression shows the positive correlation r 2 = 0.6785. The P value was

    Article Snippet: To extract the cells in sputum, fresh sputum samples were treated with 2 ml of sputolysin reagent (Calbiochem; Merck, Darmstadt, Germany) and 18 ml of phosphate-buffered saline (PBS) for 15 min at room temperature (RT) with occasional mixing.

    Techniques: Centrifugation, Enzyme-linked Immunosorbent Assay