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: 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: 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: 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: Diagnostics

Article Title: Diffusion-Tensor-Tractography-Based Diagnosis for Injury of Corticospinal Tract in a Patient with Hemiplegia Following Traumatic Brain Injury

doi: 10.3390/diagnostics10030156

Figure Lengend Snippet: Diffusion Tensor Tractography images four weeks after onset. ( A ) Tract 1 which used ROIs on the lower pons and middle pons showed that (a) the left CST was thinner than the right CST (white arrow head) and (b) discontinuation in the cerebral cortex in the left CST appeared (white dashed circle). ( B ) Tract 2 using ROIs on the lower pons and upper pons indicated the thinner left CST (a) compared with than the right CST (b) did not reach the cerebral cortex (white dashed circle) and the disconnection below the lower pons is shown (white arrow). ( C ) The disruption of the left CST around the EDH location (dashed circle) (a,b) and the dissociation of the left CST below the lower pons (white arrow). (c) Injury lesions analyzed by DTT results. Injury lesions were below the lower pons and between upper and lower pons. CST: corticospinal tract, EDH: epidural hematoma, ROI: region of interest, DTT: diffusion tensor tractography.

Article Snippet: The result of DTT using the different regions of interest (ROIs) showed that most of the right corticospinal tract (CST) did not reach the cerebral cortex around where the EDH was located, and when the ROI was placed on upper pons, a disconnection of the CST was shown and a connection of the CST in ROI with the middle pons appeared.

Techniques: Diffusion-based Assay