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: 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: FEMS Microbiology Letters

Article Title: Autophagy participates in the unfolded protein response in Toxoplasma gondii

doi: 10.1093/femsle/fnx153

Figure Lengend Snippet: Modulation of TgIF2α phosphorylation and subsequent inhibition of protein synthesis by compounds triggering ER stress and by nutrient starvation. ( A ) Schematic representation of the eIF2α pathway. ( B ) Immunoblot analysis of protein extracts from freshly egressed T. gondii tachyzoites kept extracellular for 6 hours in HBSS (for amino acids starvation), in DMEM or in DMEM supplemented with various compounds triggering ER stress: 5 mM dithiothreitol (DTT), 10 μM brefeldin A (BFA) and 10 μg/ml tunicamycin (Tuni). Total or phosphorylated TgIF2α were revealed with specific antibodies. The displayed immunoblots are representative of three independent experiments. The table below shows densitometry analysis that was carried out with values collected from the three experiments: phosphorylated TgIF2α vs total TgIF2α ratio was calculated and values were set to 1 for the DMEM control in each independent experiment, relative enrichment was then calculated for the different experimental conditions. Values displayed are mean ± standard error of the mean (SEM). ( C ) Extracellular parasites were subjected to ER stress treatment or starvation as described above, in the presence of 12.5 μM L-azidohomoalanine (AHA) for 5 hours to label newly synthesized proteins in each sample. Click reaction was performed and fluorescent protein labeling was visualized in gel (left). Coomassie staining of total proteins is shown as a control (right). Protein synthesis inhibitor cycloheximide (CHX) was used as a negative control. ( D ) Whole parasite AHA fluorescence was quantified and analyzed by flow cytometry. Values are mean from n = 3 experiments ± SEM. Asterisks denote statistical significance as determined by Student's T test.

Article Snippet: Freshly egressed parasites were collected by centrifugation and incubated for 6 hours in Hank's Balanced Salt Solution (HBSS, Gibco), or DMEM in the presence of ER stress-inducing agents: 5 mM dithiothreitol (DTT), 10 μM brefeldin A (BFA) and 10 μg/ml tunicamycin (Tuni) (all purchased from Sigma-Aldrich, Saint Quentin Fallavier, France).

Techniques: Inhibition, Western Blot, Synthesized, Labeling, Staining, Negative Control, Fluorescence, Flow Cytometry, Cytometry

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: 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

Journal: PLoS ONE

Article Title: The Dynamic of the Splicing of bZIP60 and the Proteins Encoded by the Spliced and Unspliced mRNAs Reveals Some Unique Features during the Activation of UPR in Arabidopsis thaliana

doi: 10.1371/journal.pone.0122936

Figure Lengend Snippet: Inhibition of the proteasome activity leads to the detection of GFP-bZIP60u on the ER. Seedlings (7-days-old) of transgenic Arabidopsis plants harbouring the construct 60pro:GFP-bZIP60 were pretreated with DMSO (0.2% v/v) or MG132 (100 mM) during 18 hours. Then seedlings were treated with DMSO (0.2% v/v) (A-D), DMSO (0.2% v/v) plus DTT (2 mM) (E-H), MG132 (100 μM) (I-L) or MG132 (100 μM) plus DTT (2 mM) (M-P) during 3 hours. All treatments were performed in 0.5x MS phytagel plates supplemented with the chemicals mentioned above at the indicated concentrations. After treatment, seedlings were stained with ER-Tracker Blue-White DPX (1 μM) during 45 minutes, rinsed and stained with FM 4–64 (5 μM) during additional 5 minutes at room temperature. Finally, roots were analyzed by confocal microscopy. Scale bar equals 5 μm. Images are representative of three independent experiments. (Q) Immunoblot analysis of total protein extracts obtained from bzip60 / 60pro:GFP-bZIP60 transgenic Arabidopsis seedlings (7-days-old) treated with DMSO (0.2% v/v), MG132 (100 μM) or MG132 (100 μM) plus DTT (2 mM) as mentioned above, using a polyclonal antibody against GFP. bzip60 mutant seedlings were used as control. Anti-PEPC antibody was used as loading control. Data are representative of three independent experiments. (R) CE-LIF analysis of RT-PCR products obtained from total RNA extracted from roots of bzip60 mutant or bzip60 / 60pro:GFP-bZIP60 transgenic Arabidopsis plants (7-days-old) treated with DMSO (0.2% v/v), DMSO (0.2% v/v) plus DTT (2 mM), MG132 (100 μM) or MG132 (100 μM) plus DTT (2 mM) as described above. Spherograms peaks, corresponding to the unspliced form of bZIP60 (U) and spliced form (S), are depicted. Asterisks indicate the detection of a third peak only present in DTT treated samples. Data are representative of three independent experiments.

Article Snippet: For subcellular imaging, seedlings of 7 days-old treated with DMSO, DMSO plus DTT, MG132 or MG132 plus DTT were stained with ER-Tracker Blue-White DPX dye (Molecular Probes, Life Technologies) diluted on liquid MS media during 45 minutes, rinsed on liquid MS media and stained with FM 4–64 dye (Molecular Probes, Life Technologies) during 5 minutes; at room temperature.

Techniques: Inhibition, Activity Assay, Transgenic Assay, Construct, Mass Spectrometry, Staining, Confocal Microscopy, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

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: 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

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

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

Journal: Disease Models & Mechanisms

Article Title: SILAC-based quantitative proteomics using mass spectrometry quantifies endoplasmic reticulum stress in whole HeLa cells

doi: 10.1242/dmm.040741

Figure Lengend Snippet: SILAC-based quantitative proteomics workflow. (A) Experimental design for the analyses of untreated (Ctr), DMSO-treated (Vehicle, Veh) and stressor-treated samples. HeLa cells grown in medium containing natural (light) amino acids (represented to the right) were treated with increasing concentrations of tunicamycin (in representative shades of blue). To compare the effects of different stressors, cells were also treated with thapsigargin (represented in green) and DTT (orange). A mixture of tunicamycin-treated and untreated HeLa cells labeled via SILAC with heavy lysine and arginine served as an internal standard (represented in red, see text and Materials and Methods section). The light lysates and heavy standards were mixed in a 1:1 ratio, processed by FASP and digested with trypsin. The resulting peptides were analyzed by high-resolution MS and raw files processed using the MaxQuant environment. (B) Western blot analysis with antibodies specific for known ER stress markers of the lysates used for MS analysis. Biological replicate 1, probed with antibodies specific for calnexin (CANX), ERO1-like protein alpha (ERO1A) and DNA damage-inducible transcript 3 protein (DDIT3, CHOP) and biological replicate 2, probed with anti-HSPA5 (BiP), are shown. Two different blots are shown with respective loading controls. (C) Dynamic range of the quantified proteome. The light intensity (L, see Table S1 ) of all proteins quantified by SILAC ratio is shown. The enriched categories with highest score (determined by Fisher's exact test using false discovery rate, FDR, at 0.04) and their respective P -value are shown for proteins of high (quartile 1, red), medium (quartiles 2 and 3, orange and light green) and low intensity (quartile 4, dark green). (D) Scatter plot of 2D annotation enrichment showing differences between the proteomes of DMSO- and tunicamycin (5 μg/ml)-treated cells. The experiments were performed in three independent biological replicates and each data point was analyzed in technical triplicates. The calculation of significance is described in the Materials and Methods section. Gene Ontology Biological Process (GOBP), Gene Ontology Cellular Component (GOCC), UniProt Keywords and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were analyzed (see legend).

Article Snippet: Tunicamycin and DTT were purchased from Sigma-Aldrich, thapsigargin from SERVA Electrophoresis.

Techniques: Labeling, Mass Spectrometry, Western Blot

Journal: Disease Models & Mechanisms

Article Title: SILAC-based quantitative proteomics using mass spectrometry quantifies endoplasmic reticulum stress in whole HeLa cells

doi: 10.1242/dmm.040741

Figure Lengend Snippet: Effects of different stressors on UPR activation at the proteome level. (A) Time course and dose-dependence of different stressors. HeLa cells were treated for 5 and 18 h as indicated with low (DTT 0.2 µM, thapsigargin 0.1 µm, tunicamycin 0.625 µg/ml) and high dose (DTT 2 mM, thapsigargin 1 µm, tunicamycin 5 µg/ml) of stressors. Western blot analysis shows the expression of CHOP (DDIT3) and HSPA5. Tubulin and Ponceau S are shown as loading controls. The experiment was carried out in three replicates. The HSPA5:tubulin ratio ( N =3, mean±s.d.), quantified using ImageJ, is shown as a bar graph. Controls at 18 h are shown. (B) Principal component analysis (PCA) comparing the proteome remodeling induced by tunicamycin (5 μg/ml), DTT (2 mM) and thapsigargin (1 µM). Vehicle-treated cells were used as controls. Each treatment was performed in biological triplicates. Data were filtered for 100% valid values in ANOVA-significant proteins (543 proteins). (C) Loadings of B, showing the major separators into components one and two ( x - and y -axis, respectively). TIMP1, metalloproteinase inhibitor 1; HSPA5, endoplasmic reticulum chaperone BiP; IFRD1, interferon-related developmental regulator 1; GPRC5A, retinoic acid-induced protein 3; SCD, stearoyl-CoA desaturase 5; PON2, serum paraoxonase/arylesterase 2; KIAA0101, PCNA-associated factor (also known as PCLAF); TK1, thymidine kinase. (D) Unsupervised clustering of ANOVA-significant proteins showing three main clusters, with profiles and corresponding relative enrichments, indicated to the right. Enrichment is based on Fisher's exact test of keyword annotations at 0.04 FDR.

Article Snippet: Tunicamycin and DTT were purchased from Sigma-Aldrich, thapsigargin from SERVA Electrophoresis.

Techniques: Activation Assay, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Assembly and Folding Properties of Cytosolic IgG Intrabodies

doi: 10.1038/s41598-020-58798-7

Figure Lengend Snippet: IgG1s expressed in the reducing cytosol and via the oxidizing ER assemble differently. ( a ) The concentration of free thiols in PBS was measured before and after DTT removal steps. ( b ) Immunoblotting analysis of the assembly pattern of H and L chains following DTT removal. HEK293 cells transfected with KV10ΔLd-3D8 IgG or KV10Ld-3D8 IgG. Lysates of transfectants were treated with 100 mM DTT for 30 min at room temperature, followed by DTT removal steps comprising passage through a desalting column and dialysis against PBS at 4 °C for 24 hours. Lysates were separated by non-reducing SDS-PAGE, and the H chain was detected by immunoblotting with anti-IgG/Fc antibody.

Article Snippet: To validate the complete removal of DTT in the above column and dialysis steps, 100 mM DTT in PBS was passed through the desalting column then subjected to dialysis against PBS, and the concentration of DTT was determined using a free thiol detection kit (Abcam; cat# ab112158).

Techniques: Concentration Assay, Transfection, SDS Page

Journal: Carbohydrate polymers

Article Title: Hyaluronate Coating Enhances the Delivery and Biocompatibility of Gold Nanoparticles

doi: 10.1016/j.carbpol.2018.01.046

Figure Lengend Snippet: HS-HA synthesis chemistry. In the presence of borate buffer at pH9, the terminal cyclic hemiacetal ring opens and changes to a linear aldehyde form. A Schiff base forms with cystamine. Thus, the terminal saccharide reverses between two states to reach the final form. The Amadori compound, sodium cyanoborohydride (NaBH 3 CN), is added, and then Dithiothreitol (DTT) is added to reduce the disulfide and produce HS -HAs.

Article Snippet: Dithiothreitol (DTT) was obtained from Duchefa Biochemie (Haarlem, Netherlands).

Techniques:

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: International Journal of Molecular Sciences

Article Title: Preparation of Biodegradable Oligo(lactide)s-Grafted Dextran Nanogels for Efficient Drug Delivery by Controlling Intracellular Traffic

doi: 10.3390/ijms19061606

Figure Lengend Snippet: Photographs of EI 4 /Gal-Dex- g -SS-OLA nanogel before and after 24 h incubation in the presence of 10 mM dithiothreitol (DTT) in PB solution at 25 °C with ( right ) and without ( left ) laser beam irradiation.

Article Snippet: N ,N ’-Carbonyldiimidazole (CDI), 4-(N ,N -dimethylamino)-pyridine (DMAP), di-tert -butyl dicarbonate, cystamine dihydrochloride, dithiothreitol (DTT), and lactose were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan).

Techniques: Incubation, Irradiation

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