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  • 99
    Thermo Fisher dtt
    The NPXY motif and GULP are required for internalization of Jedi-1. Time course for internalization of Jedi-1-GFP (A) or Jedi-1-GFP APXA mutant (B) after addition of microspheres. HeLa cells were transfected with wild-type or APXA mutant Jedi-1-GFP. Surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin at 4°C and then exposed to fluorescent microspheres and warmed to 37°C for the time indicated. The <t>biotinylation</t> of surface proteins was then reversed using the reducing agent <t>DTT.</t> The cells were lysed, and internalized, biotinylated Jedi-1 or mutant Jedi-1 was pulled down with avidin-conjugated agarose beads and detected by immunoblotting with a GFP antibody. Total levels of Jedi-1 are shown in lysates ( n = 3). (C) Control MEFs or MEFs with GULP knocked down (MEF psiGULP) were transfected with Jedi-GFP. The cells were then exposed to microspheres or left untreated for 90 min, and the internalized Jedi-1 was detected as in A and B. Levels of Jedi-1–GFP and GULP are shown in the lysates.
    Dtt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 31062 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    IBI Scientific dithiothreitol dtt cleland s reagent
    The NPXY motif and GULP are required for internalization of Jedi-1. Time course for internalization of Jedi-1-GFP (A) or Jedi-1-GFP APXA mutant (B) after addition of microspheres. HeLa cells were transfected with wild-type or APXA mutant Jedi-1-GFP. Surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin at 4°C and then exposed to fluorescent microspheres and warmed to 37°C for the time indicated. The <t>biotinylation</t> of surface proteins was then reversed using the reducing agent <t>DTT.</t> The cells were lysed, and internalized, biotinylated Jedi-1 or mutant Jedi-1 was pulled down with avidin-conjugated agarose beads and detected by immunoblotting with a GFP antibody. Total levels of Jedi-1 are shown in lysates ( n = 3). (C) Control MEFs or MEFs with GULP knocked down (MEF psiGULP) were transfected with Jedi-GFP. The cells were then exposed to microspheres or left untreated for 90 min, and the internalized Jedi-1 was detected as in A and B. Levels of Jedi-1–GFP and GULP are shown in the lysates.
    Dithiothreitol Dtt Cleland S Reagent, supplied by IBI Scientific, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dithiothreitol dtt cleland s reagent/product/IBI Scientific
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    99
    Bio-Rad dithiothreitol
    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>
    Dithiothreitol, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 2165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dithiothreitol/product/Bio-Rad
    Average 99 stars, based on 2165 article reviews
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    Thermo Fisher dithiotreitol dtt
    The reducing agent <t>dithiothreitol</t> <t>(DTT)</t> causes large voltage offsets in Ag/AgCl electrodes. Summary of the voltage offsets produced by 1 mM DTT. Two wire purities of 99.9 and 99.99% silver were examined. As in , bare wire ( A ), which simulates the
    Dithiotreitol Dtt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Abcam dithiothreitol dtt
    The reducing agent <t>dithiothreitol</t> <t>(DTT)</t> causes large voltage offsets in Ag/AgCl electrodes. Summary of the voltage offsets produced by 1 mM DTT. Two wire purities of 99.9 and 99.99% silver were examined. As in , bare wire ( A ), which simulates the
    Dithiothreitol Dtt, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc dithiothreitol dtt
    The reducing agent <t>dithiothreitol</t> <t>(DTT)</t> causes large voltage offsets in Ag/AgCl electrodes. Summary of the voltage offsets produced by 1 mM DTT. Two wire purities of 99.9 and 99.99% silver were examined. As in , bare wire ( A ), which simulates the
    Dithiothreitol Dtt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The NPXY motif and GULP are required for internalization of Jedi-1. Time course for internalization of Jedi-1-GFP (A) or Jedi-1-GFP APXA mutant (B) after addition of microspheres. HeLa cells were transfected with wild-type or APXA mutant Jedi-1-GFP. Surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin at 4°C and then exposed to fluorescent microspheres and warmed to 37°C for the time indicated. The biotinylation of surface proteins was then reversed using the reducing agent DTT. The cells were lysed, and internalized, biotinylated Jedi-1 or mutant Jedi-1 was pulled down with avidin-conjugated agarose beads and detected by immunoblotting with a GFP antibody. Total levels of Jedi-1 are shown in lysates ( n = 3). (C) Control MEFs or MEFs with GULP knocked down (MEF psiGULP) were transfected with Jedi-GFP. The cells were then exposed to microspheres or left untreated for 90 min, and the internalized Jedi-1 was detected as in A and B. Levels of Jedi-1–GFP and GULP are shown in the lysates.

    Journal: Molecular Biology of the Cell

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism

    doi: 10.1091/mbc.E13-11-0658

    Figure Lengend Snippet: The NPXY motif and GULP are required for internalization of Jedi-1. Time course for internalization of Jedi-1-GFP (A) or Jedi-1-GFP APXA mutant (B) after addition of microspheres. HeLa cells were transfected with wild-type or APXA mutant Jedi-1-GFP. Surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin at 4°C and then exposed to fluorescent microspheres and warmed to 37°C for the time indicated. The biotinylation of surface proteins was then reversed using the reducing agent DTT. The cells were lysed, and internalized, biotinylated Jedi-1 or mutant Jedi-1 was pulled down with avidin-conjugated agarose beads and detected by immunoblotting with a GFP antibody. Total levels of Jedi-1 are shown in lysates ( n = 3). (C) Control MEFs or MEFs with GULP knocked down (MEF psiGULP) were transfected with Jedi-GFP. The cells were then exposed to microspheres or left untreated for 90 min, and the internalized Jedi-1 was detected as in A and B. Levels of Jedi-1–GFP and GULP are shown in the lysates.

    Article Snippet: The biotinylation of surface proteins was reversed using 50 mM DTT at various time points after stimulation with 2-μm carboxylate-modified latex beads (Invitrogen).

    Techniques: Mutagenesis, Transfection, Avidin-Biotin Assay

    Antibody conjugation of nanoparticles with polysiloxane copolymer coating. Abbreviations: PEO, poly(ethylene oxide); IONPs, iron oxide nanoparticles; APTES, (3-aminopropyl) triethoxysilane; Sulfo-LC-SPDP, Sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate; DTT, dithiothreitol.

    Journal: International Journal of Nanomedicine

    Article Title: Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

    doi: 10.2147/IJN.S49069

    Figure Lengend Snippet: Antibody conjugation of nanoparticles with polysiloxane copolymer coating. Abbreviations: PEO, poly(ethylene oxide); IONPs, iron oxide nanoparticles; APTES, (3-aminopropyl) triethoxysilane; Sulfo-LC-SPDP, Sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate; DTT, dithiothreitol.

    Article Snippet: Materials Sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (Sulfo-LC-SPDP), and dithiothreitol were purchased from Pierce Biotechnology (Rockford IL, USA).

    Techniques: Conjugation Assay

    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

    FasL induces caspase-dependent cleavage of Grx1 and increases PSSG as well as S-glutathionylation of Fas. (A) Immunoblot analysis of cleaved caspase-8 (p18) and -3 fragments (p17 and p19) in C10 cells treated with FasL + M2 as described in Fig. 1 in the presence or absence of 10 µM ZVAD-FMK. The bottom panel shows total cellular content of Grx1. Note that expression of the pro form of caspase-8 remains unchanged during the course of the experiment. (B) Evaluation of the interaction between Grx1 and caspase-8 or -3 in cells. C10 cells were exposed to FasL + M2 as described in Fig. 1 A , and Grx1 was immunoprecipitated (IP) at the indicated times for the evaluation of association with active caspase-8 or -3 fragments via Western blotting. The bottom panel represents a Grx1 immunoblot. Lanes on the right represent lysates from cells treated with FasL + M2 for 2 h but were subjected to IgG IP as a reagent control. All samples were run on the same gel, and the lanes were cut and reassembled for consistency. The bottom panels represent total content of proteins in whole cell lysates (WCL) that were used as the input for IP. Note that expression of the pro form of caspase-8 remains unchanged during the course of the experiment. Black line indicates that intervening lanes have been spliced out. (C) In vitro assessment of cleavage of Grx1 by caspase-8 or -3. 200 ng recombinant hGrx1 was incubated with 200 U active caspase-8 or -3. At the indicated times, samples were prepared for immunoblot analysis of hGrx1. Fragmented hGrx1 product is ∼8 kD in size. Incubation of heat-inactivated caspase-8 and -3 with hGrx1 for 4 h largely prevented the formation of cleaved fragment (0 h). (D) Increases in overall PSSG are a response to ligation of Fas and are caspase dependent. Cells were incubated as described in A. ZVAD-FMK or vehicle was added to cells 2 h before ligation of Fas as well as 2 h after ligation. Lysates were resolved by nonreducing SDS-PAGE. Antiglutathione antibody was used to detect PSSG on immunoblots. The bottom panel shows total Fas content. (E) Caspase-dependent S-glutathionylation of Fas. C10 cells were incubated with FasL + M2 for 0.5, 1, or 2 h in the presence or absence of ZVAD-FMK. Cell lysates were subjected to nonreducing IP (−DTT) using antiglutathione antibody to IP S-glutathionylated proteins (IP: PSSG) before detection of Fas via Western blotting. As a reagent control to reduce S-glutathionylated proteins before IP, samples were incubated with 50 mM DTT (+DTT). The bottom panel represents Fas content in cell lysates. (F) S-glutathionylation of Fas requires the presence of caspase-8. C10 cells were transfected with control (Ctr) siRNA or caspase-8 (C8)–specific siRNA and 48 h later were incubated with FasL + M2 for 2 or 4 h. The top lane shows assessment of S-glutathionylation of Fas via IP of S-glutathionylated proteins using antiglutathione antibody (IP: PSSG) under nonreducing conditions (−DTT) before detection of Fas via Western blotting. As a reagent control to reduce S-glutathionylated proteins before IP, samples were incubated with 50 mM DTT (+DTT). The bottom panels show total content of Fas, procaspase-8, cleaved caspase-8, cleaved caspase-3, and Grx1 in whole cell lysates. (G) Assessment of caspase-dependent degradation of Grx1 and S-glutathionylation of Fas in NIH 3T3 cells after ligation of Fas. Cells were treated with 500 ng/ml FasL + 1 µg/ml M2 for 1, 2, or 4 h in the presence or absence of ZVAD-FMK. S-glutathionylated proteins were immunoprecipitated as described in E before detection of Fas via Western blotting. The bottom panel represents Fas content, cleaved caspase-3, and Grx1 content in whole cell lysates.

    Journal: The Journal of Cell Biology

    Article Title: Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas

    doi: 10.1083/jcb.200807019

    Figure Lengend Snippet: FasL induces caspase-dependent cleavage of Grx1 and increases PSSG as well as S-glutathionylation of Fas. (A) Immunoblot analysis of cleaved caspase-8 (p18) and -3 fragments (p17 and p19) in C10 cells treated with FasL + M2 as described in Fig. 1 in the presence or absence of 10 µM ZVAD-FMK. The bottom panel shows total cellular content of Grx1. Note that expression of the pro form of caspase-8 remains unchanged during the course of the experiment. (B) Evaluation of the interaction between Grx1 and caspase-8 or -3 in cells. C10 cells were exposed to FasL + M2 as described in Fig. 1 A , and Grx1 was immunoprecipitated (IP) at the indicated times for the evaluation of association with active caspase-8 or -3 fragments via Western blotting. The bottom panel represents a Grx1 immunoblot. Lanes on the right represent lysates from cells treated with FasL + M2 for 2 h but were subjected to IgG IP as a reagent control. All samples were run on the same gel, and the lanes were cut and reassembled for consistency. The bottom panels represent total content of proteins in whole cell lysates (WCL) that were used as the input for IP. Note that expression of the pro form of caspase-8 remains unchanged during the course of the experiment. Black line indicates that intervening lanes have been spliced out. (C) In vitro assessment of cleavage of Grx1 by caspase-8 or -3. 200 ng recombinant hGrx1 was incubated with 200 U active caspase-8 or -3. At the indicated times, samples were prepared for immunoblot analysis of hGrx1. Fragmented hGrx1 product is ∼8 kD in size. Incubation of heat-inactivated caspase-8 and -3 with hGrx1 for 4 h largely prevented the formation of cleaved fragment (0 h). (D) Increases in overall PSSG are a response to ligation of Fas and are caspase dependent. Cells were incubated as described in A. ZVAD-FMK or vehicle was added to cells 2 h before ligation of Fas as well as 2 h after ligation. Lysates were resolved by nonreducing SDS-PAGE. Antiglutathione antibody was used to detect PSSG on immunoblots. The bottom panel shows total Fas content. (E) Caspase-dependent S-glutathionylation of Fas. C10 cells were incubated with FasL + M2 for 0.5, 1, or 2 h in the presence or absence of ZVAD-FMK. Cell lysates were subjected to nonreducing IP (−DTT) using antiglutathione antibody to IP S-glutathionylated proteins (IP: PSSG) before detection of Fas via Western blotting. As a reagent control to reduce S-glutathionylated proteins before IP, samples were incubated with 50 mM DTT (+DTT). The bottom panel represents Fas content in cell lysates. (F) S-glutathionylation of Fas requires the presence of caspase-8. C10 cells were transfected with control (Ctr) siRNA or caspase-8 (C8)–specific siRNA and 48 h later were incubated with FasL + M2 for 2 or 4 h. The top lane shows assessment of S-glutathionylation of Fas via IP of S-glutathionylated proteins using antiglutathione antibody (IP: PSSG) under nonreducing conditions (−DTT) before detection of Fas via Western blotting. As a reagent control to reduce S-glutathionylated proteins before IP, samples were incubated with 50 mM DTT (+DTT). The bottom panels show total content of Fas, procaspase-8, cleaved caspase-8, cleaved caspase-3, and Grx1 in whole cell lysates. (G) Assessment of caspase-dependent degradation of Grx1 and S-glutathionylation of Fas in NIH 3T3 cells after ligation of Fas. Cells were treated with 500 ng/ml FasL + 1 µg/ml M2 for 1, 2, or 4 h in the presence or absence of ZVAD-FMK. S-glutathionylated proteins were immunoprecipitated as described in E before detection of Fas via Western blotting. The bottom panel represents Fas content, cleaved caspase-3, and Grx1 content in whole cell lysates.

    Article Snippet: As a control, a portion of the lysate was treated with 50 mM DTT to reduce glutathionylated proteins, and these samples were purified through columns (Micro-BioSpin; Bio-Rad Laboratories) to remove DTT before subsequent IP.

    Techniques: Expressing, Immunoprecipitation, Western Blot, In Vitro, Recombinant, Incubation, Ligation, SDS Page, Transfection

    Increased S-glutathionylation of Fas, caspase-8 activity, and cell death in cells lacking Grx1. (A) Assessment of S-glutathionylation of Fas after knockdown of Grx1. C10 cells were transfected with Grx1 siRNA or control (Ctr) siRNA and treated with FasL + M2 for the indicated times. S-glutathionylated proteins were immunoprecipitated using antiglutathione antibody (IP: PSSG). Samples treated with 50 mM DTT to reduce S-glutathionylated proteins (+DTT) were used as reagent controls. The content of Fas, Grx1, and actin in whole cell lysates (WCL) used as the input for IP are shown in the bottom panels. (B) Assessment of S-glutathionylation of Fas after loading of cells with biotinylated glutathione. siRNA-transfected cells were labeled with 5 mM biotinylated glutathione ethyl ester for 1 h before treatment with FasL. After 2 h of FasL + M2 treatment, glutathionylated proteins in lysates were immunoprecipitated using antibiotin antibody followed by immunoblot detection of Fas. The bottom panel shows total Fas expression in whole cell lysates as a loading control. (C) Evaluation of caspase-8 and -3 enzymatic activities in cells after knockdown of Grx1. C10 cells were transfected with control or Grx1 siRNA before stimulation with FasL + M2 for 1 h, and lysates were prepared for evaluation of caspase activity. Results are expressed as mean + SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas

    doi: 10.1083/jcb.200807019

    Figure Lengend Snippet: Increased S-glutathionylation of Fas, caspase-8 activity, and cell death in cells lacking Grx1. (A) Assessment of S-glutathionylation of Fas after knockdown of Grx1. C10 cells were transfected with Grx1 siRNA or control (Ctr) siRNA and treated with FasL + M2 for the indicated times. S-glutathionylated proteins were immunoprecipitated using antiglutathione antibody (IP: PSSG). Samples treated with 50 mM DTT to reduce S-glutathionylated proteins (+DTT) were used as reagent controls. The content of Fas, Grx1, and actin in whole cell lysates (WCL) used as the input for IP are shown in the bottom panels. (B) Assessment of S-glutathionylation of Fas after loading of cells with biotinylated glutathione. siRNA-transfected cells were labeled with 5 mM biotinylated glutathione ethyl ester for 1 h before treatment with FasL. After 2 h of FasL + M2 treatment, glutathionylated proteins in lysates were immunoprecipitated using antibiotin antibody followed by immunoblot detection of Fas. The bottom panel shows total Fas expression in whole cell lysates as a loading control. (C) Evaluation of caspase-8 and -3 enzymatic activities in cells after knockdown of Grx1. C10 cells were transfected with control or Grx1 siRNA before stimulation with FasL + M2 for 1 h, and lysates were prepared for evaluation of caspase activity. Results are expressed as mean + SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P

    Article Snippet: As a control, a portion of the lysate was treated with 50 mM DTT to reduce glutathionylated proteins, and these samples were purified through columns (Micro-BioSpin; Bio-Rad Laboratories) to remove DTT before subsequent IP.

    Techniques: Activity Assay, Transfection, Immunoprecipitation, Labeling, Expressing

    Overexpression of Grx1 decreases S-glutathionylation of Fas, caspase-8 and -3 activities, and cell death. (A) Assessment of Grx1 expression. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids (1 µg/4 × 10 5 cells). Cells were stimulated with FasL + M2 for 2 h, and lysates were analyzed by immunoblotting for Grx1 expression. The bottom panel shows β-actin as a loading control. (B) Assessment of S-glutathionylation of Fas after overexpression of Grx1. pcDNA3 or Flag-Grx1–transfected cells were stimulated with FasL + M2, and after 2 h, S-glutathionylated proteins were immunoprecipitated using an antibody directed against glutathione (IP: PSSG). +DTT samples were used as reagent controls. The bottom panel represents Fas content in whole cell lysates (WCL). (C) Assessment of caspase-8 and -3 activities in cells after overexpression of Grx1. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids and stimulated with FasL + M2. At the indicated times, cells were harvested, and lysates were prepared for evaluation of caspase activity. Results are expressed as mean ± SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas

    doi: 10.1083/jcb.200807019

    Figure Lengend Snippet: Overexpression of Grx1 decreases S-glutathionylation of Fas, caspase-8 and -3 activities, and cell death. (A) Assessment of Grx1 expression. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids (1 µg/4 × 10 5 cells). Cells were stimulated with FasL + M2 for 2 h, and lysates were analyzed by immunoblotting for Grx1 expression. The bottom panel shows β-actin as a loading control. (B) Assessment of S-glutathionylation of Fas after overexpression of Grx1. pcDNA3 or Flag-Grx1–transfected cells were stimulated with FasL + M2, and after 2 h, S-glutathionylated proteins were immunoprecipitated using an antibody directed against glutathione (IP: PSSG). +DTT samples were used as reagent controls. The bottom panel represents Fas content in whole cell lysates (WCL). (C) Assessment of caspase-8 and -3 activities in cells after overexpression of Grx1. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids and stimulated with FasL + M2. At the indicated times, cells were harvested, and lysates were prepared for evaluation of caspase activity. Results are expressed as mean ± SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P

    Article Snippet: As a control, a portion of the lysate was treated with 50 mM DTT to reduce glutathionylated proteins, and these samples were purified through columns (Micro-BioSpin; Bio-Rad Laboratories) to remove DTT before subsequent IP.

    Techniques: Over Expression, Expressing, Transfection, Immunoprecipitation, Activity Assay

    Iron-sulfur cluster transfer from SufBC 2 D to AcnB. Holo-SufBC 2 D complex (0.3 nmol), [Fe-S] ( gray bars ), or [Fe-S] + FADH 2 ( hatched bars ), was co-incubated in 10 μl of 50 m m Tris-HCl, pH 7.6, with ( a ) and without ( b ) 5 m m DTT with apo-AcnB (0.2

    Journal: The Journal of Biological Chemistry

    Article Title: Iron-Sulfur (Fe-S) Cluster Assembly

    doi: 10.1074/jbc.M110.127449

    Figure Lengend Snippet: Iron-sulfur cluster transfer from SufBC 2 D to AcnB. Holo-SufBC 2 D complex (0.3 nmol), [Fe-S] ( gray bars ), or [Fe-S] + FADH 2 ( hatched bars ), was co-incubated in 10 μl of 50 m m Tris-HCl, pH 7.6, with ( a ) and without ( b ) 5 m m DTT with apo-AcnB (0.2

    Article Snippet: For the experiment in the absence of DTT during the FeS transfer, apo-aconitase B was first pretreated with 5 m m DTT for 30 min, before desalting the protein solution via a MicroBiospin column (Bio-Rad).

    Techniques: Incubation

    Subcloned and purified anti-β-sheet conformational monoclonal antibody (aβComAb) GW-23B7-specific reactivity to human paired helical filaments (PHFs) and oligomeric Aβ peptides. a Enzyme-linked immunosorbent assay (ELISA) data showing the reactivity from cell supernatant of subcloned GW-23B7 immunoglobulin M (IgM) and an irrelevant clone from the same fusion to PHFs and oligomers differential on Aβ 1–40 and Aβ 1–42 . The right panel shows the even coating of the selected peptides on the plate and the lack of unspecific reactivity to secondary antimouse IgM. b Western blots showing the pentameric integrity of the purified aβComAb GW-23B7. Lane 1 = unreduced sample, lane 2 = reduced with 0.1 M dithiothreitol (DTT). Left panel : Fast Green reversible protein stain. Middle panel : Antimouse IgM (μ-specific) light chain. Right panel : Antimouse kappa light chain. IgMp Pentameric immunoglobulin M, Hμr μ heavy chain reduced, Kr Kappa light chain reduced. c ELISA results showing the reactivity of purified aβComAb GW-23B7 diluted 1:1000 to Aβ 1–40 , Aβ 1–42 , and human PHFs. d Surface plasmon resonance showing the binding affinity of the purified aβComAb GW-23B7 to the oligomeric species of Aβ 1–42 and the lack of binding affinity to the monomeric forms. The dissociation constant (K D ) (14 nM) was determined from the raw data on the left

    Journal: Alzheimer's Research & Therapy

    Article Title: Anti-β-sheet conformation monoclonal antibody reduces tau and Aβ oligomer pathology in an Alzheimer’s disease model

    doi: 10.1186/s13195-018-0337-3

    Figure Lengend Snippet: Subcloned and purified anti-β-sheet conformational monoclonal antibody (aβComAb) GW-23B7-specific reactivity to human paired helical filaments (PHFs) and oligomeric Aβ peptides. a Enzyme-linked immunosorbent assay (ELISA) data showing the reactivity from cell supernatant of subcloned GW-23B7 immunoglobulin M (IgM) and an irrelevant clone from the same fusion to PHFs and oligomers differential on Aβ 1–40 and Aβ 1–42 . The right panel shows the even coating of the selected peptides on the plate and the lack of unspecific reactivity to secondary antimouse IgM. b Western blots showing the pentameric integrity of the purified aβComAb GW-23B7. Lane 1 = unreduced sample, lane 2 = reduced with 0.1 M dithiothreitol (DTT). Left panel : Fast Green reversible protein stain. Middle panel : Antimouse IgM (μ-specific) light chain. Right panel : Antimouse kappa light chain. IgMp Pentameric immunoglobulin M, Hμr μ heavy chain reduced, Kr Kappa light chain reduced. c ELISA results showing the reactivity of purified aβComAb GW-23B7 diluted 1:1000 to Aβ 1–40 , Aβ 1–42 , and human PHFs. d Surface plasmon resonance showing the binding affinity of the purified aβComAb GW-23B7 to the oligomeric species of Aβ 1–42 and the lack of binding affinity to the monomeric forms. The dissociation constant (K D ) (14 nM) was determined from the raw data on the left

    Article Snippet: For electrophoresis to confirm the identity of aβComAb GW-23B7, 1 μg of antibody with or without dithiothreitol (DTT) 0.1 M was mixed with an equal volume of tricine sample buffer (Bio-Rad Laboratories, Hercules, CA, USA), electrophoresed on Bolt™ 4–12% Bis-Tris (Thermo Fisher Scientific) polyacrylamide gels and system, and transferred onto nitrocellulose membranes (NCs) for 1 h at 386 mA in 0.1% 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) (Sigma-Aldrich, St. Louis, MO, USA)-10% methanol.

    Techniques: Purification, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, SPR Assay, Binding Assay

    The reducing agent dithiothreitol (DTT) causes large voltage offsets in Ag/AgCl electrodes. Summary of the voltage offsets produced by 1 mM DTT. Two wire purities of 99.9 and 99.99% silver were examined. As in , bare wire ( A ), which simulates the

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Redox artifacts in electrophysiological recordings

    doi: 10.1152/ajpcell.00318.2012

    Figure Lengend Snippet: The reducing agent dithiothreitol (DTT) causes large voltage offsets in Ag/AgCl electrodes. Summary of the voltage offsets produced by 1 mM DTT. Two wire purities of 99.9 and 99.99% silver were examined. As in , bare wire ( A ), which simulates the

    Article Snippet: Tris-2-carboxyethly-phosphine (TCEP) and dithiothreitol (DTT) were obtained from Pierce.

    Techniques: Produced