Journal: PLoS ONE

Article Title: Inhibition of Helicobacter pylori and Its Associated Urease by Palmatine: Investigation on the Potential Mechanism

doi: 10.1371/journal.pone.0168944

Figure Lengend Snippet: Reversal of Pal-inhibited HPU (A) and JBU (B) activity by DTT. The urease enzymatic activity was inhibited by Pal (•), and DTT (▪) was added post 20 min. In the assay system, 1.25 mM DTT and 0.5 mM Pal were used for both ureases (0.25 mg/mL). Activity of Pal-inhibited urease was monitored before and after DTT addition, 1.25 mM DTT without was used as a control. Results summarized here are the mean value of n = 3 ± SD.

Article Snippet: Acetohydroxamic acid (AHA, CAS number: 546-88-3, purity: 98%), urea (Molecular Biology Reagent), jack bean urease (JBU, type III with specific activity 40.3 U/mg solid), HEPES (Amresco > 99%), L-cysteine (L-cys), glutathione (GSH), dithiothreithol (DTT), boric acid (BA) and sodium fluoride (NaF) were all purchased from Sigma-Aldrich (St Louis, MO, USA).

Techniques: Activity Assay

Journal: Open Biology

Article Title: Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells

doi: 10.1098/rsob.160098

Figure Lengend Snippet: NCC is the main form of injury-induced protein aggregation. ( a – d ) Jurkat cells were treated with vehicle (uninjured), etoposide, staurosporine or Fas ligand for 24 h. ( a ) Triton-soluble and -insoluble proteins were isolated and subjected to SDS-PAGE under reducing conditions followed by immunoblot analysis. Results are representative of n = 3–6 independent experiments. ( b ) Triton-soluble and -insoluble proteins were isolated and subjected to SDS-PAGE in the presence/absence of dithiothreitol (DTT) followed by immunoblot analysis. Results are representative of n = 2–3 independent experiments. ( c ) Cells were fixed, subjected to immunofluorescence and imaged by super-resolution microscopy with xy- resolution of 70 nm. Shown are representative x , y micrographs, where the bottom two rows are magnified micrographs taken from the boxed areas in the top two rows. Staurosporine-induced necrosis causes NCC-proteins (α-tubulin, EF2 and MCM7) to relocate into aberrant and distinct structures. Arrows indicate EF2 deposits that exhibit little-to-no staining for tubulin or MCM7. Arrowheads indicate MCM7 deposits that exhibit little-to-no staining for tubulin or EF2. Note, the procedure used to stain oxidized thiols was not compatible with the sample preparation for super-resolution microscopy. ( d ) Cells were fixed and subjected to immunofluorescence with maleimide co-staining for oxidized thiols. Shown are representative x,y confocal micrographs. Staurosporine-induced necrosis leads to nuclear breakdown and a marked increase in oxidized thiols that colocalize with aberrantly deposited NCC-proteins (3PDGH, GAPDH, MCM7, EF1, EF2 and α-tubulin).

Article Snippet: SDS-PAGE for tryptic digestion Samples were boiled in 2% SDS-loading buffer with dithiothreitol and subjected to SDS-PAGE using a NuPAGE Novex Bis-Tris Mini gel (4–12%) with NuPAGE MOPS SDS-Running buffer supplemented with 1× NuPAGE antioxidant (Life Technologies).

Techniques: Isolation, SDS Page, Immunofluorescence, Microscopy, Staining, Sample Prep

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

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

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

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

Journal: PLoS Pathogens

Article Title: SnTox3 Acts in Effector Triggered Susceptibility to Induce Disease on Wheat Carrying the Snn3 Gene

doi: 10.1371/journal.ppat.1000581

Figure Lengend Snippet: SnTox3 activity is sensitive to Dithiothreitol (DTT). Leaves of BG220 were infiltrated with culture filtrates of P. pastoris expressing SnTox3 that had been treated for 2 h with 0 mM, 5 mM and 10 mM DTT.

Article Snippet: To investigate if the predicted disulfide bonds were important for toxin activity, the P. pastoris culture containing SnTox3 was treated with Dithiothreitol (DTT) (Fisher Scientific, Pittsburgh, PA) at two different concentrations including 5 mM and 10 mM, with water added as a negative control.

Techniques: Activity Assay, Expressing

Journal: PLoS Pathogens

Article Title: Oxidative stress and protein damage responses mediate artemisinin resistance in malaria parasites

doi: 10.1371/journal.ppat.1006930

Figure Lengend Snippet: Chemosensitivity profiling of in vitro -selected P . falciparum . Chemosensitivity phenotypes of our artemisinin-resistant parasites were evaluated. (A) Apart from their ring-stage sensitivity, the artemisinin IC50 4hr for the trophozoite (IC50 20hpi/4hr ) and schizont (IC50 30hpi/4hr ) stages was also measured in all parasite lines. Differential susceptibility to artemisinin between selected and control parasites, was subsequently validated using a standard in vitro (72-hour) drug assay (IC50) (B) , and the Ring stage Survival Assay (RSA) (C) . Additional data can be found in Figs 2A and S2B and S2 Table . (D) Ring-stage susceptibility (IC50 10hpi/4hr ) against a pulse exposure to artemisinin derivatives Dihydroartemisinin (DHA) and Artesunate (ATS) were also monitored, while chemosensitivity against non-artemisinin antimalarials, quinine (QN), chloroquine (CQ), mefloquine (MEF) and pyrimethamine (PYR), were evaluated using a standard drug assay (IC50). Additional data can be found in S3A and S3B Fig and S3 Table . (E) Apart from antimalarial drugs, the ring-stage sensitivity of all parasite lines was also measured for compounds that are related to the mechanism of action of artemisinin: hydrogen peroxide (H 2 O 2 ), dithiothreitol (DTT), and epoxomicin (EPX). Additional data can be found in S3C Fig and S3 Table . All drug assays were performed in biological triplicates; error bars represent the standard deviation. Pairwise comparison of percent survival (RSA), IC50 10hpi/4hr and IC50 values between resistant and sensitive parasites was performed using student’s t-test.

Article Snippet: For artesunate (ATS) (Sigma), dihydroartemisinin (DHA) (Sigma), dithiothreitol (DTT) (Merck), epoxomicin (EPX) (Sigma) and hydrogen peroxide (H2 O2 ) (Merck), parasites were incubated in drug for 4 hours at 10–14 HPI, after which the cultures were washed twice with fresh media to remove the drug, and finally resuspended at 1% hematocrit and then allowed to reinvade.

Techniques: In Vitro, Clonogenic Cell Survival Assay, Standard Deviation

Journal: Nanoscale Research Letters

Article Title: Biocleavable Polycationic Micelles as Highly Efficient Gene Delivery Vectors

doi: 10.1007/s11671-010-9716-9

Figure Lengend Snippet: Agarose gel electrophoresis images of polycationic micelles/pEGFP-C1 complexes pretreated with various concentrations of dithiothreitol (DTT) solutions for different time. Polyplexes at N/P ratio of 20. Lane 0: naked DNA; lane 1–5: DTT concentration of 0, 1.0, 2.5, 5.0, 10 mM

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

Techniques: Agarose Gel Electrophoresis, Concentration Assay

Journal: Nature Communications

Article Title: Self-interaction of NPM1 modulates multiple mechanisms of liquid–liquid phase separation

doi: 10.1038/s41467-018-03255-3

Figure Lengend Snippet: Only NPM1 constructs with an intact IDR undergo homotypic LLPS. a Phase diagrams for NPM1 WT , NPM1 N240 and NPM1 N188 in buffer comprised of 10 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5 in the presence of 150 mg/mL Ficoll PM70. The purple dotted line is a visual aid that represents the phase boundary for NPM1 WT between the mixed (gray circles) and demixed (green circles) states. Representative microscopy images are shown for 20 µM protein samples. Scale bar = 5 µm; b confocal microscopy images of homotypic NPM1 droplets at 20 µM NPM1 in the presence of 150 mg/mL Ficoll PM70, in buffers containing the indicated NaCl concentration. Scale bar = 10 µm

Article Snippet: The affinity tags were removed in an overnight dialysis step at 4 °C, against 4 L 10 mM Tris pH 7.5, 150 mM NaCl, 2 mM DTT buffer, in the presence of TEV or HRV3C (BioVision, Milpitas, CA) protease.

Techniques: Construct, Microscopy, Confocal Microscopy, Concentration Assay

Journal: Nature Communications

Article Title: Self-interaction of NPM1 modulates multiple mechanisms of liquid–liquid phase separation

doi: 10.1038/s41467-018-03255-3

Figure Lengend Snippet: Two-dimensional size-and-shape distribution analyses of the sedimentation velocity AUC data. a NPM1, b NPM1 N240 , c NPM1 N188 , and d NPM1 CTD . The transformed velocity data are shown as contour plots (heat maps) of c ( M, f/f 0 , ) (top), c ( s, f/f 0 ) (middle), and c ( s, M ) (bottom) with 0 fringes/S (white) to maximum value fringes/S (red), with increasing color temperature indicating higher values. Velocity data were acquired at 50,000 rpm at 20 °C in buffer comprised of 10 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT. The s w , ( f/f 0 ) w , and M- values are listed in Supplementary Table 2

Article Snippet: The affinity tags were removed in an overnight dialysis step at 4 °C, against 4 L 10 mM Tris pH 7.5, 150 mM NaCl, 2 mM DTT buffer, in the presence of TEV or HRV3C (BioVision, Milpitas, CA) protease.

Techniques: Sedimentation, Transformation Assay

Journal: Molecular Vision

Article Title: Bilberry extract and anthocyanins suppress unfolded protein response induced by exposure to blue LED light of cells in photoreceptor cell line

doi:

Figure Lengend Snippet: Effect of bilberry extract, and Dp3G, Cy3G, or Mv3G individually on DTT-induced cell death in murine photoreceptor cell line. A : Experimental protocol in vitro. Effect of ( B ) bilberry extract, ( C ) delphinidin-3-glucoside (Dp3G), ( D ) cyanidin-3-glucoside (Cy3G), and ( E ) malvidin-3-glucoside (Mv3G) on dithiothreitol (DTT)-induced cell death in murine photoreceptor cells. The number of cells exhibiting propidium iodide (PI) fluorescence was counted and expressed as a percentage of Hoechst 33342–positive cells. Data are the means ± standard error of the mean (SEM; n=5 to 6). ## p

Article Snippet: Dithiothreitol (DTT) was purchased from Wako Pure Chemical Industries, Ltd. Osaka, Japan.

Techniques: In Vitro, Fluorescence