dithiothreitol  (Millipore)


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    L Dithiothreitol
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    Catalog Number:
    d9760
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    Applications:
    L-DTT [(2R,3R)-1,4-dimercapto-2,3-butanediol; L-dithiothreitol], a chiral bidentate dithiol with two stereogenic centers, may be used in chiroptical response research. L-DDT is proposed as a stereoselective reducing agent for disulfide bridges in complex molecules.
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    Images

    1) Product Images from "ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway"

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

    Journal: Cell

    doi: 10.1016/j.cell.2014.06.026

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

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

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

    Techniques Used: Expressing, Autoradiography

    2) Product Images from "Mon1 Is Essential for Fungal Virulence and Stress Survival in Cryptococcus neoformans"

    Article Title: Mon1 Is Essential for Fungal Virulence and Stress Survival in Cryptococcus neoformans

    Journal: Mycobiology

    doi: 10.1080/12298093.2018.1468053

    Phenotypes of the Cn mon1 Δ mutant following exposure to various stresses. Spot dilution assays with WT (H99), Cn cna1 Δ (KK1), Cn mon1 Δ (HP55 and HP56), and Cn mon1 + Cn MON1 (HPC3 and HPC4). Cells were incubated overnight, diluted 10-fold, and plated on YPD agar. Plated cells were incubated for 2 days at 30, 37, 38, and 39 °C. (A) Serially diluted cells were also plated on YPD agar without or with (B) dithiothreitol (DTT), (C) sodium dodecylsulfate (SDS), (D) calcofluor white, (E) congo red, and (F) fluconazole at the indicated concentrations. Results shown are representative of two independent experiments.
    Figure Legend Snippet: Phenotypes of the Cn mon1 Δ mutant following exposure to various stresses. Spot dilution assays with WT (H99), Cn cna1 Δ (KK1), Cn mon1 Δ (HP55 and HP56), and Cn mon1 + Cn MON1 (HPC3 and HPC4). Cells were incubated overnight, diluted 10-fold, and plated on YPD agar. Plated cells were incubated for 2 days at 30, 37, 38, and 39 °C. (A) Serially diluted cells were also plated on YPD agar without or with (B) dithiothreitol (DTT), (C) sodium dodecylsulfate (SDS), (D) calcofluor white, (E) congo red, and (F) fluconazole at the indicated concentrations. Results shown are representative of two independent experiments.

    Techniques Used: Mutagenesis, Incubation

    3) Product Images from "The Rho Exchange Factors Vav2 and Vav3 Favor Skin Tumor Initiation and Promotion by Engaging Extracellular Signaling LoopsVav Proteins' Role in Skin Cancer"

    Article Title: The Rho Exchange Factors Vav2 and Vav3 Favor Skin Tumor Initiation and Promotion by Engaging Extracellular Signaling LoopsVav Proteins' Role in Skin Cancer

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001615

    Vav proteins participate in the initiation phase of skin tumors. (A) Scheme of these experiments. (B, C) Example (B; scale bar, 100 µm) and quantification (C) of apoptotic cells found in the epidermis of mice of the indicated genotypes 24 h upon the application of DMBA ( n = 4). In (B), sections were stained with antibodies to the cleaved fragment of caspase 3 (ID number: 12367) and 14, 4′,6-diamidino-2-phenylindole (DAPI) to reveal apoptotic cells (red color) and cell nuclei (blue color), respectively. (D, E) Example (D; scale bar, 100 µm) and quantification (E) of phospho-histone H2AX + keratinocytes (brown color) present in the epidermis of short-term DMBA-treated mice of indicated genotypes ( n = 4). p-, phosphorylated. (F) Number of apoptotic (annexin V + ) wild-type and Vav2 −/− ; Vav3 −/− keratinocytes induced after 8 (left panel) and 12 (right panel) h in the indicated culture conditions ( n = 4). Bleo, bleomycin; DTT, dithiothreitol; NS, no statistically significant. (G, H) Example of a flow cytometry experiment (G) and subsequent quantification (H) of the level of apoptosis induced by either DMBA (G, H) or serum starvation (H) in wild-type and Vav2 −/− ; Vav3 −/− keratinocytes ectopically expressing GFP either alone (G, H) or in combination with HA-Vav2 (H) or Myc-Vav3 (G, H) ( n = 3). In (G), only the gated GFP + cells are shown.
    Figure Legend Snippet: Vav proteins participate in the initiation phase of skin tumors. (A) Scheme of these experiments. (B, C) Example (B; scale bar, 100 µm) and quantification (C) of apoptotic cells found in the epidermis of mice of the indicated genotypes 24 h upon the application of DMBA ( n = 4). In (B), sections were stained with antibodies to the cleaved fragment of caspase 3 (ID number: 12367) and 14, 4′,6-diamidino-2-phenylindole (DAPI) to reveal apoptotic cells (red color) and cell nuclei (blue color), respectively. (D, E) Example (D; scale bar, 100 µm) and quantification (E) of phospho-histone H2AX + keratinocytes (brown color) present in the epidermis of short-term DMBA-treated mice of indicated genotypes ( n = 4). p-, phosphorylated. (F) Number of apoptotic (annexin V + ) wild-type and Vav2 −/− ; Vav3 −/− keratinocytes induced after 8 (left panel) and 12 (right panel) h in the indicated culture conditions ( n = 4). Bleo, bleomycin; DTT, dithiothreitol; NS, no statistically significant. (G, H) Example of a flow cytometry experiment (G) and subsequent quantification (H) of the level of apoptosis induced by either DMBA (G, H) or serum starvation (H) in wild-type and Vav2 −/− ; Vav3 −/− keratinocytes ectopically expressing GFP either alone (G, H) or in combination with HA-Vav2 (H) or Myc-Vav3 (G, H) ( n = 3). In (G), only the gated GFP + cells are shown.

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing

    4) Product Images from "Propolis Modulates Fibronectin Expression in the Matrix of Thermal Injury"

    Article Title: Propolis Modulates Fibronectin Expression in the Matrix of Thermal Injury

    Journal: BioMed Research International

    doi: 10.1155/2014/748101

    The characteristics of molecular profile of extracts differently treated burn wounds buffered by urea solution and sodium dodecyl sulfate (SDS). Extract components, subjected to dithiothreitol as a factor reducing the disulfide bonds, were subjected to the electrophoresis in a 6% polyacrylamide gel in the presence of SDS. Lane 1, molecular mass markers; lane 2, standard of collagen type I; lanes 3, 4, 5, and 6, components extracted, on the 5th day of healing, from wounds treated by NaCl, SSD, propolis, and propolis vehicle, respectively. The arrows indicate the migration position of standards of known molecular weights (60 kDa, 100 kDa, and 220 kDa).
    Figure Legend Snippet: The characteristics of molecular profile of extracts differently treated burn wounds buffered by urea solution and sodium dodecyl sulfate (SDS). Extract components, subjected to dithiothreitol as a factor reducing the disulfide bonds, were subjected to the electrophoresis in a 6% polyacrylamide gel in the presence of SDS. Lane 1, molecular mass markers; lane 2, standard of collagen type I; lanes 3, 4, 5, and 6, components extracted, on the 5th day of healing, from wounds treated by NaCl, SSD, propolis, and propolis vehicle, respectively. The arrows indicate the migration position of standards of known molecular weights (60 kDa, 100 kDa, and 220 kDa).

    Techniques Used: Electrophoresis, Migration

    5) Product Images from "Functions of MutL?, Replication Protein A (RPA), and HMGB1 in 5?-Directed Mismatch Repair *"

    Article Title: Functions of MutL?, Replication Protein A (RPA), and HMGB1 in 5?-Directed Mismatch Repair *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.021287

    Effects of RPA, MutLα, and HMGB1 on the processive action of MutSα-activated Exo1. Protein challenge was used to evaluate effects of repair activities on processive hydrolysis by activated Exo1. A , reactions contained (per time sample) 24 fmol of gapped homoduplex DNA substrate (gap size 314 nucleotides) and 400 fmol of MutSα. After 5 min at 37 °C, hydrolysis was initiated at time 0 by addition of 21 fmol of Exo1 in the absence (buffer, left ) or presence of either 280 fmol of MutLα ( middle ) or 3.3 pmol of HMGB1 ( right ). Reaction conditions were the same as those used for mismatch-provoked excision (“Experimental Procedures”) except that the volume per time sample was reduced to 18.4 μl to accommodate the further addition at 1 min of 1.6 μl buffer (16 m m HEPES-KOH pH 7.5, 100 m m KCl, 0.3 m m dithiothreitol, 1 mg/ml BSA, 5% (v/v) glycerol). Reactions were sampled (20 μl), hydrolysis quenched, and products visualized after cleavage with ClaI, alkaline-agarose gel electrophoresis, Southern transfer, and hybridization with probe V2532 (“Experimental Procedures”). B , reaction conditions were as in panel A , except that hydrolysis was initiated by addition of Exo1 only. At 1 min, reactions were supplemented per time sample with 900 fmol of RPA ( left ), 280 fmol of MutLα ( middle ), or 3.3 pmol of HMGB1 ( right ) in 1.6 μl of buffer.
    Figure Legend Snippet: Effects of RPA, MutLα, and HMGB1 on the processive action of MutSα-activated Exo1. Protein challenge was used to evaluate effects of repair activities on processive hydrolysis by activated Exo1. A , reactions contained (per time sample) 24 fmol of gapped homoduplex DNA substrate (gap size 314 nucleotides) and 400 fmol of MutSα. After 5 min at 37 °C, hydrolysis was initiated at time 0 by addition of 21 fmol of Exo1 in the absence (buffer, left ) or presence of either 280 fmol of MutLα ( middle ) or 3.3 pmol of HMGB1 ( right ). Reaction conditions were the same as those used for mismatch-provoked excision (“Experimental Procedures”) except that the volume per time sample was reduced to 18.4 μl to accommodate the further addition at 1 min of 1.6 μl buffer (16 m m HEPES-KOH pH 7.5, 100 m m KCl, 0.3 m m dithiothreitol, 1 mg/ml BSA, 5% (v/v) glycerol). Reactions were sampled (20 μl), hydrolysis quenched, and products visualized after cleavage with ClaI, alkaline-agarose gel electrophoresis, Southern transfer, and hybridization with probe V2532 (“Experimental Procedures”). B , reaction conditions were as in panel A , except that hydrolysis was initiated by addition of Exo1 only. At 1 min, reactions were supplemented per time sample with 900 fmol of RPA ( left ), 280 fmol of MutLα ( middle ), or 3.3 pmol of HMGB1 ( right ) in 1.6 μl of buffer.

    Techniques Used: Recombinase Polymerase Amplification, Agarose Gel Electrophoresis, Hybridization

    6) Product Images from "IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila"

    Article Title: IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila

    Journal: mBio

    doi: 10.1128/mBio.02629-19

    Differential effect of UPR stress stimuli on rescue of L. pneumophila replication in IFN-γ-stimulated macrophages. (A) Log 10 RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated for 48 h postinfection with 6.0 ng/ml IFN-γ, 2.0 mM 2DG, 2.0 μM geldanamycin (geld.), 1.0 μg/ml brefeldin A (BfA), and 2.0 mM dithiothreitol (DTT) as indicated. (B) Log 10 RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated for 48 h postinfection with 6.0 ng/ml IFN-γ, 10.0 μM tunicamycin (tunic.), and 25.0 nM thapsigargin (thaps.) as indicated. (C) Log 10 RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated for 48 h postinfection with 6.0 ng/ml IFN-γ, 2.0 mM 2DG, 2.0 μM geldanamycin, and 0.1 μM ISRIB as indicated. ***, P
    Figure Legend Snippet: Differential effect of UPR stress stimuli on rescue of L. pneumophila replication in IFN-γ-stimulated macrophages. (A) Log 10 RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated for 48 h postinfection with 6.0 ng/ml IFN-γ, 2.0 mM 2DG, 2.0 μM geldanamycin (geld.), 1.0 μg/ml brefeldin A (BfA), and 2.0 mM dithiothreitol (DTT) as indicated. (B) Log 10 RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated for 48 h postinfection with 6.0 ng/ml IFN-γ, 10.0 μM tunicamycin (tunic.), and 25.0 nM thapsigargin (thaps.) as indicated. (C) Log 10 RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated for 48 h postinfection with 6.0 ng/ml IFN-γ, 2.0 mM 2DG, 2.0 μM geldanamycin, and 0.1 μM ISRIB as indicated. ***, P

    Techniques Used: Infection

    7) Product Images from "ATORVASTATIN OR TRANSGENIC EXPRESSION OF TFPI INHIBITS COAGULATION INITIATED BY ANTI-NONGAL IgG BINDING TO PORCINE AORTIC ENDOTHELIAL CELLS"

    Article Title: ATORVASTATIN OR TRANSGENIC EXPRESSION OF TFPI INHIBITS COAGULATION INITIATED BY ANTI-NONGAL IgG BINDING TO PORCINE AORTIC ENDOTHELIAL CELLS

    Journal: Journal of thrombosis and haemostasis : JTH

    doi: 10.1111/j.1538-7836.2010.03950.x

    Sensitized baboon serum anti-nonGal Abs activate PAECs to induce TF activity through IgG binding A. Heat-inactivated sensitized baboon serum (HI SBS) was pre-incubated with dithiothreitol (DTT, 200μl/ml) to eliminate IgM. IgM or IgG binding to PAECs was determined by flow cytometry. (Shaded: isotype control; solid line: non-treated serum; broken line: serum treated with DTT). B. WT PAECs were incubated with medium (control) or HI SBS, which has been pretreated with DTT for 30min. TF activity on the surface of PAECs was activated by HI SBS was unrelated to the pretreatment of DTT ( the level of IgM) by recalcified clotting assay. C. The experiment performed in B was repeated with HI SBS that had been treated with an anti-IgG Fab Ab at various concentrations (5–20μg/ml) for 30min at 4°C. Anti-IgG Fab Ab blocked the effect of HI SBS (with high levels of anti-nonGal Abs), inactivating WT PAECS (* p
    Figure Legend Snippet: Sensitized baboon serum anti-nonGal Abs activate PAECs to induce TF activity through IgG binding A. Heat-inactivated sensitized baboon serum (HI SBS) was pre-incubated with dithiothreitol (DTT, 200μl/ml) to eliminate IgM. IgM or IgG binding to PAECs was determined by flow cytometry. (Shaded: isotype control; solid line: non-treated serum; broken line: serum treated with DTT). B. WT PAECs were incubated with medium (control) or HI SBS, which has been pretreated with DTT for 30min. TF activity on the surface of PAECs was activated by HI SBS was unrelated to the pretreatment of DTT ( the level of IgM) by recalcified clotting assay. C. The experiment performed in B was repeated with HI SBS that had been treated with an anti-IgG Fab Ab at various concentrations (5–20μg/ml) for 30min at 4°C. Anti-IgG Fab Ab blocked the effect of HI SBS (with high levels of anti-nonGal Abs), inactivating WT PAECS (* p

    Techniques Used: Activity Assay, Binding Assay, Incubation, Flow Cytometry, Cytometry, Coagulation

    8) Product Images from "Mutations in L-type amino acid transporter-2 support SLC7A8 as a novel gene involved in age-related hearing loss"

    Article Title: Mutations in L-type amino acid transporter-2 support SLC7A8 as a novel gene involved in age-related hearing loss

    Journal: eLife

    doi: 10.7554/eLife.31511

    Hearing phenotype of C57BL6/J-129Sv Slc7a8 knockout mice. ( A ) Representative image of western bloting of total membranes from kidney, brain and intestine of wild-type (+/+) and Slc7a8 knock out (-/-) mice in the absence (-) or presence (+) of 100 mM dithiothreitol reducing agent (DTT) of three independent biological samples for both sexes (male and female). Protein (50 μg) were loaded in 7% acrylamide SDS-PAGE gel. Molecular mass standard (KDa) are indicated. Red arrows point SLC7A8/CD98hc heterodimer band as well as the light subunit SLC7A8. Upper panel: Rabbit anti-SLC7A8. Bottom panel: Mouse anti-βactin. ( B ) Pre-Pulse Inhibition of the acoustic startle response (PPI). Mean and SEM are represented. Pulse: 120 dB single pulse. Pre-pulse inhibition test: six different pre-pulse intensities (70 to 90 dB) in pseudo random order with 15 s inter-trial intervals. Wild type (white circles, n = 19) and Slc7a8 −/− (blue circles, n = 15) from 4- to 7-month-old are represented. Significant differences were determined using paired Student’s t-test, ***p
    Figure Legend Snippet: Hearing phenotype of C57BL6/J-129Sv Slc7a8 knockout mice. ( A ) Representative image of western bloting of total membranes from kidney, brain and intestine of wild-type (+/+) and Slc7a8 knock out (-/-) mice in the absence (-) or presence (+) of 100 mM dithiothreitol reducing agent (DTT) of three independent biological samples for both sexes (male and female). Protein (50 μg) were loaded in 7% acrylamide SDS-PAGE gel. Molecular mass standard (KDa) are indicated. Red arrows point SLC7A8/CD98hc heterodimer band as well as the light subunit SLC7A8. Upper panel: Rabbit anti-SLC7A8. Bottom panel: Mouse anti-βactin. ( B ) Pre-Pulse Inhibition of the acoustic startle response (PPI). Mean and SEM are represented. Pulse: 120 dB single pulse. Pre-pulse inhibition test: six different pre-pulse intensities (70 to 90 dB) in pseudo random order with 15 s inter-trial intervals. Wild type (white circles, n = 19) and Slc7a8 −/− (blue circles, n = 15) from 4- to 7-month-old are represented. Significant differences were determined using paired Student’s t-test, ***p

    Techniques Used: Knock-Out, Mouse Assay, Western Blot, SDS Page, Inhibition

    9) Product Images from "Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody"

    Article Title: Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody

    Journal: Toxins

    doi: 10.3390/toxins8040100

    Sequential [ 32 P]-ADP-ribosylation of Rho. CHO cells ( a ) and HT22 cells ( c ) were treated with different C3 concentrations for indicated time points. Subsequently, cell lysates were incubated with 1 µM C3 and 1 µCi [ 32 P]-NAD in 20 µL of buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.3), 10 mM MgCl 2 , 10 mM dithiothreitol, 10 mM thymidine, and 10 µM nicotinamide adenine dinucleotide (NAD) at 37 °C for 30 min. The reaction was terminated by addition of Laemmli sample buffer and then incubated at 95 °C for 10 min. Samples were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the [ 32 P]-ADP-ribosylated Rho was analyzed by phosphorimaging. Representative phosphorimaging of [ 32 P]-ADP-ribosylated Rho and the Coomassie brilliant blue stained SDS-PAGE gels as loading control are presented ( n = 4). Molecular masses are indicated in kDa. M = Marker SDS7 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany); and ( b , d ) diagrams depict cellular level of unmodified Rho (● control, ■ 10 nM C3, ▲ 100 nM C3, ★ 1000 nM C3). Cellular level of unmodified Rho was determined by densitometrically quantification of intensity of sequential [ 32 P]-ADP-ribosylated Rho using Kodak software 1D, Version 3.5. The signal intensity of [ 32 P]-ADP-ribosylated Rho from untreated control cells were set as 1. Statistical differences were determined by two-sided Student’s t test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
    Figure Legend Snippet: Sequential [ 32 P]-ADP-ribosylation of Rho. CHO cells ( a ) and HT22 cells ( c ) were treated with different C3 concentrations for indicated time points. Subsequently, cell lysates were incubated with 1 µM C3 and 1 µCi [ 32 P]-NAD in 20 µL of buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.3), 10 mM MgCl 2 , 10 mM dithiothreitol, 10 mM thymidine, and 10 µM nicotinamide adenine dinucleotide (NAD) at 37 °C for 30 min. The reaction was terminated by addition of Laemmli sample buffer and then incubated at 95 °C for 10 min. Samples were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the [ 32 P]-ADP-ribosylated Rho was analyzed by phosphorimaging. Representative phosphorimaging of [ 32 P]-ADP-ribosylated Rho and the Coomassie brilliant blue stained SDS-PAGE gels as loading control are presented ( n = 4). Molecular masses are indicated in kDa. M = Marker SDS7 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany); and ( b , d ) diagrams depict cellular level of unmodified Rho (● control, ■ 10 nM C3, ▲ 100 nM C3, ★ 1000 nM C3). Cellular level of unmodified Rho was determined by densitometrically quantification of intensity of sequential [ 32 P]-ADP-ribosylated Rho using Kodak software 1D, Version 3.5. The signal intensity of [ 32 P]-ADP-ribosylated Rho from untreated control cells were set as 1. Statistical differences were determined by two-sided Student’s t test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Marker, Software

    10) Product Images from "Cyclotides Isolated from an Ipecac Root Extract Antagonize the Corticotropin Releasing Factor Type 1 Receptor"

    Article Title: Cyclotides Isolated from an Ipecac Root Extract Antagonize the Corticotropin Releasing Factor Type 1 Receptor

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00616

    MALDI-TOF mass spectrometry (MS) of crude and chemically modified cyclotide-enriched extract. Mass spectra of (A) unmodified extract, (B) reduced and alkylated crude extract (red.+alk.) using dithiothreitol and iodoacetamide, and (C) endo-GluC digested extract are shown. Masses labeled in the spectra refer to monoisotopic [M+H] + and cyclotides are labeled according to Table 1 . Using this analysis workflow, circular peptides containing six cysteines typically exhibit a mass shift of +348 Da (red.+alk.) and +366 Da (endo-GluC).
    Figure Legend Snippet: MALDI-TOF mass spectrometry (MS) of crude and chemically modified cyclotide-enriched extract. Mass spectra of (A) unmodified extract, (B) reduced and alkylated crude extract (red.+alk.) using dithiothreitol and iodoacetamide, and (C) endo-GluC digested extract are shown. Masses labeled in the spectra refer to monoisotopic [M+H] + and cyclotides are labeled according to Table 1 . Using this analysis workflow, circular peptides containing six cysteines typically exhibit a mass shift of +348 Da (red.+alk.) and +366 Da (endo-GluC).

    Techniques Used: Mass Spectrometry, Modification, Labeling

    11) Product Images from "Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in Candida lusitaniae"

    Article Title: Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in Candida lusitaniae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044192

    Calcineurin mutation confers cell wall integrity defects in C. lusitaniae . ( A ) Calcineurin mutants are sensitive to cell wall integrity-damaging agents and ER stress chemicals. Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium containing sodium dodecyl sulfate (SDS), calcofluor white (CFW), congo red (CR), tunicamycin (TM), or dithiothreitol (DTT) and incubated at 30°C for 48 h. ( B ) The growth kinetics of C. lusitaniae wild-type and mutant strains at 30°C. Cells were grown overnight at 30°C, washed twice with dH 2 O, diluted to 0.2 OD 600 /ml in fresh liquid YPD medium, and incubated at 30°C with shaking at 250 rpm. The OD 600 of cultures was measured at 0, 3, 6, 9, 12, 15, 18, 21, and 24 h. The experiments were performed in triplicate, and data was plotted using Prism 5.03. Strains tested were wild-type (ATCC42720), cnb1 mutants (YC198 and YC202), and crz1 mutants (YC187 and YC467).
    Figure Legend Snippet: Calcineurin mutation confers cell wall integrity defects in C. lusitaniae . ( A ) Calcineurin mutants are sensitive to cell wall integrity-damaging agents and ER stress chemicals. Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium containing sodium dodecyl sulfate (SDS), calcofluor white (CFW), congo red (CR), tunicamycin (TM), or dithiothreitol (DTT) and incubated at 30°C for 48 h. ( B ) The growth kinetics of C. lusitaniae wild-type and mutant strains at 30°C. Cells were grown overnight at 30°C, washed twice with dH 2 O, diluted to 0.2 OD 600 /ml in fresh liquid YPD medium, and incubated at 30°C with shaking at 250 rpm. The OD 600 of cultures was measured at 0, 3, 6, 9, 12, 15, 18, 21, and 24 h. The experiments were performed in triplicate, and data was plotted using Prism 5.03. Strains tested were wild-type (ATCC42720), cnb1 mutants (YC198 and YC202), and crz1 mutants (YC187 and YC467).

    Techniques Used: Mutagenesis, Incubation

    12) Product Images from "Dyclonine rescues frataxin deficiency in animal models and buccal cells of patients with Friedreich's ataxia"

    Article Title: Dyclonine rescues frataxin deficiency in animal models and buccal cells of patients with Friedreich's ataxia

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddu408

    High-throughput screening reveals that dyclonine protects FA patient fibroblasts from diamide stress. ( A ) Effect of antioxidant inhibitors on 50B11 cell viability with FXN knockdown. Eleven inhibitors of thiol-related antioxidants were tested in an siRNA-mediated, FXN-deficient 50B11 DRG cell line [10 µM antimycin A, 1 µM auranofin, 100 µM BSO, 100 µM carmustine, 10 µM diamide, 0.1% diethyl maleate (DEM), 0.1% ethanol, 0.03% H 2 O 2 , 1 m m l -glutathione ( l -GSH), 0.1% phenethyl isothiocyanate (PEITC), 100 µM dichloronitrobenzene (DCNB) and 1 µM N -methyl protoporphyrin (NMP)]. Cell viability was measured with Calcein-AM after 24 h and normalized to untreated control ( n = 3). Increased sensitivity to cell death was induced by inhibitors of thiol-related antioxidants diamide and auranofin in FXN knockdown cells compared with AllStars non-targeting siRNA negative control. ( B ) FXN-dependent sensitivity to diamide is dose-dependent in 50B11 cells. Cell viability was measured with Calcein-AM after 24 h of treatment with 3–300 µM diamide and normalized to untreated control ( n = 3). ( C ) Friedreich's patient cells are sensitive to diamide. To confirm these effects in patient cells with low FXN, we tested 100 µM diamide in fibroblasts and lymphoblasts and found that patient cells were more sensitive to diamide compared with healthy control cells. ( D ) Results of high-throughput screen for drugs that protect from diamide toxicity. Significance is shown comparing healthy volunteer and FA patient lines grouped together. This cell-based assay in FA patient fibroblast cell line 1134 was further optimized for high-throughput screening in 96-well plates, with a mean Z ′-value of 0.75 ( n = 25). This platform was used to screen a library of 1600 drugs that have been approved for clinical use. FA fibroblasts were pretreated with 10 µM test compound, DMSO (negative control) or 300 µM dithiothreitol (DTT) (positive control) for 24 h and followed by 100 µM diamide for 24 h. Cell viability was measured with Calcein-AM. Screening data (diamide + all drugs) are the mean of two replicates and presented as fold above DMSO + diamide control. Arrow indicates dyclonine response. Mean + SD for the 1600 drugs was 1 ± 0.3; mean + SEM was 1 ± 0.01. Compounds that rescued from diamide toxicity greater than mean + 2× SD advanced to secondary screening, which included replication of protective effect in a concentration-dependent manner, 0.01–10 µM. ( E ) An example of dose-dependent protection by dyclonine. Dyclonine was added to FA fibroblast line 1134 for 24 h before 100 µM diamide treatment, and Calcein-AM viability is shown as fold above DMSO + diamide control. Intrinsic effect: 2.1 ± 0.49-fold above DMSO + diamide control; EC 50 : 0.36 ± 0.25 μM ( n = 3). ( F ) Chemical structure of dyclonine. The plotted data in A, B, C and E display mean responses and error bars represent SD ( n = 3–4). *P
    Figure Legend Snippet: High-throughput screening reveals that dyclonine protects FA patient fibroblasts from diamide stress. ( A ) Effect of antioxidant inhibitors on 50B11 cell viability with FXN knockdown. Eleven inhibitors of thiol-related antioxidants were tested in an siRNA-mediated, FXN-deficient 50B11 DRG cell line [10 µM antimycin A, 1 µM auranofin, 100 µM BSO, 100 µM carmustine, 10 µM diamide, 0.1% diethyl maleate (DEM), 0.1% ethanol, 0.03% H 2 O 2 , 1 m m l -glutathione ( l -GSH), 0.1% phenethyl isothiocyanate (PEITC), 100 µM dichloronitrobenzene (DCNB) and 1 µM N -methyl protoporphyrin (NMP)]. Cell viability was measured with Calcein-AM after 24 h and normalized to untreated control ( n = 3). Increased sensitivity to cell death was induced by inhibitors of thiol-related antioxidants diamide and auranofin in FXN knockdown cells compared with AllStars non-targeting siRNA negative control. ( B ) FXN-dependent sensitivity to diamide is dose-dependent in 50B11 cells. Cell viability was measured with Calcein-AM after 24 h of treatment with 3–300 µM diamide and normalized to untreated control ( n = 3). ( C ) Friedreich's patient cells are sensitive to diamide. To confirm these effects in patient cells with low FXN, we tested 100 µM diamide in fibroblasts and lymphoblasts and found that patient cells were more sensitive to diamide compared with healthy control cells. ( D ) Results of high-throughput screen for drugs that protect from diamide toxicity. Significance is shown comparing healthy volunteer and FA patient lines grouped together. This cell-based assay in FA patient fibroblast cell line 1134 was further optimized for high-throughput screening in 96-well plates, with a mean Z ′-value of 0.75 ( n = 25). This platform was used to screen a library of 1600 drugs that have been approved for clinical use. FA fibroblasts were pretreated with 10 µM test compound, DMSO (negative control) or 300 µM dithiothreitol (DTT) (positive control) for 24 h and followed by 100 µM diamide for 24 h. Cell viability was measured with Calcein-AM. Screening data (diamide + all drugs) are the mean of two replicates and presented as fold above DMSO + diamide control. Arrow indicates dyclonine response. Mean + SD for the 1600 drugs was 1 ± 0.3; mean + SEM was 1 ± 0.01. Compounds that rescued from diamide toxicity greater than mean + 2× SD advanced to secondary screening, which included replication of protective effect in a concentration-dependent manner, 0.01–10 µM. ( E ) An example of dose-dependent protection by dyclonine. Dyclonine was added to FA fibroblast line 1134 for 24 h before 100 µM diamide treatment, and Calcein-AM viability is shown as fold above DMSO + diamide control. Intrinsic effect: 2.1 ± 0.49-fold above DMSO + diamide control; EC 50 : 0.36 ± 0.25 μM ( n = 3). ( F ) Chemical structure of dyclonine. The plotted data in A, B, C and E display mean responses and error bars represent SD ( n = 3–4). *P

    Techniques Used: High Throughput Screening Assay, Negative Control, Cell Based Assay, Positive Control, Concentration Assay

    13) Product Images from "Convergent Evolution of Calcineurin Pathway Roles in Thermotolerance and Virulence in Candida glabrata"

    Article Title: Convergent Evolution of Calcineurin Pathway Roles in Thermotolerance and Virulence in Candida glabrata

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.112.002279

    Calcineurin controls ER stress tolerance, cell wall integrity, and pH homeostasis in C. glabrata . (A) Calcineurin mutants are hypersensitive to ER stress chemicals and cell wall integrity-damaging agents. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing tunicamycin (TM), dithiothreitol (DTT), calcofluor white (CFW), or Congo red (CR), and incubated at 24° for 48 hr. (B) Calcineurin and crz1 mutants are hypersensitive to the cell membrane integrity-damaging agent sodium dodecyl sulfate (SDS). Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing SDS and incubated at indicated temperatures for 48 hr. (C) Calcineurin is required for growth at acidic and alkaline environments. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing 150 mM HEPES buffered at pH 2 or 5. For pH 8 medium, YPD was buffered with 150 mM pH 9 HEPES (85 ml of YPD plus 15 ml of 1M HEPES at pH 9) due to slightly acidic YPD medium. The pHs of solid media were confirmed with pH indicator strips.
    Figure Legend Snippet: Calcineurin controls ER stress tolerance, cell wall integrity, and pH homeostasis in C. glabrata . (A) Calcineurin mutants are hypersensitive to ER stress chemicals and cell wall integrity-damaging agents. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing tunicamycin (TM), dithiothreitol (DTT), calcofluor white (CFW), or Congo red (CR), and incubated at 24° for 48 hr. (B) Calcineurin and crz1 mutants are hypersensitive to the cell membrane integrity-damaging agent sodium dodecyl sulfate (SDS). Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing SDS and incubated at indicated temperatures for 48 hr. (C) Calcineurin is required for growth at acidic and alkaline environments. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing 150 mM HEPES buffered at pH 2 or 5. For pH 8 medium, YPD was buffered with 150 mM pH 9 HEPES (85 ml of YPD plus 15 ml of 1M HEPES at pH 9) due to slightly acidic YPD medium. The pHs of solid media were confirmed with pH indicator strips.

    Techniques Used: Incubation

    14) Product Images from "Lpg0393 of Legionella pneumophila Is a Guanine-Nucleotide Exchange Factor for Rab5, Rab21 and Rab22"

    Article Title: Lpg0393 of Legionella pneumophila Is a Guanine-Nucleotide Exchange Factor for Rab5, Rab21 and Rab22

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118683

    Overall structure of Lpg0393Δ17. (A) Ribbon drawings in two orientations. The dotted line indicates a disordered segment in the crystal structure. (B) Two Lpg0393Δ17 molecules (Chain A and Chain B) forming a crystallographic dimer. The experimental SAD map together with the final refined model is shown for a region that contains a methionine residue. (C) Gel filtration analysis. Lpg0393Δ17 (5 mg/ml) was eluted from a HiLoad 26/60 Superdex 75 column at a rate of 1.5 ml/min with 30 mM TrisHCl buffer (pH 8.0) containing 100 mM NaCl and 3 mM dithiothreitol. The size marker proteins were bovine serum albumin (67 kDa), ovalbumin (43 kDa), yellow fluorescent protein (27 kDa) and chymotrypsinogen A (25 kDa).
    Figure Legend Snippet: Overall structure of Lpg0393Δ17. (A) Ribbon drawings in two orientations. The dotted line indicates a disordered segment in the crystal structure. (B) Two Lpg0393Δ17 molecules (Chain A and Chain B) forming a crystallographic dimer. The experimental SAD map together with the final refined model is shown for a region that contains a methionine residue. (C) Gel filtration analysis. Lpg0393Δ17 (5 mg/ml) was eluted from a HiLoad 26/60 Superdex 75 column at a rate of 1.5 ml/min with 30 mM TrisHCl buffer (pH 8.0) containing 100 mM NaCl and 3 mM dithiothreitol. The size marker proteins were bovine serum albumin (67 kDa), ovalbumin (43 kDa), yellow fluorescent protein (27 kDa) and chymotrypsinogen A (25 kDa).

    Techniques Used: Filtration, Marker

    15) Product Images from "Glutathione-mediated release of Bodipy® from PEG cofunctionalized gold nanoparticles"

    Article Title: Glutathione-mediated release of Bodipy® from PEG cofunctionalized gold nanoparticles

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S33726

    FL L-cystine dissociation to form monomer in presence of dithiothreitol.
    Figure Legend Snippet: FL L-cystine dissociation to form monomer in presence of dithiothreitol.

    Techniques Used:

    16) Product Images from "Calcium Efflux From the Endoplasmic Reticulum Leads to β-Cell Death"

    Article Title: Calcium Efflux From the Endoplasmic Reticulum Leads to β-Cell Death

    Journal: Endocrinology

    doi: 10.1210/en.2013-1519

    β-Cell stressors induce ER calcium depletion. A, Rates of ER calcium-depleted INS-1 832/13 cells treated with indicated concentrations of glucose for 48 hours. B and C, Rates of ER calcium-depleted cells (B) and cell death (C) in INS-1 832/13 cells treated with 25 mM glucose (HG) for the indicated periods of time. D, Rates of ER calcium-depleted cells in INS1 832/13 treated with or without 25 mM 2-deoxyglucose (2DG) together with 25 mM high glucose (HG) for 48 hours. E, Rates of ER calcium-depleted INS-1 832/13 cells untreated or treated with 0.5 mM palmitic acid for the indicated periods of time. F, Rates of ER calcium-depleted INS-1 832/13 cells untreated (BSA) or treated with 0.5 mM oleic acid (OA), 0.5 mM palmitic acid (PA), 0.5 mM palmitic acid combined with high glucose (PA+HG), or 0.5 mM palmitic acid combined with 0.5 mM oleic acid (PA+OA) for 24 hours. G, Cytosolic calcium levels in INS1 832/13 cells untreated (UT) or treated with 0.5 mM palmitic acid (PA) or 0.5 mM palmitic acid together with high glucose (PA+HG) for 24 hours. The calcium efflux from the ER was induced by 3 μM thapsigargin at time 0 (arrow), and the calcium level was determined by Fluo-4 staining. H, Rates of cell death in INS-1 832/13 cells untreated (UT) or treated with 0.5 mM palmitic acid (PA), 0.5 mM oleic acid (OA), or both (PA+OA) were determined by propidium iodide staining. I, Rates of ER calcium-depleted INS-1 832/13 cells untreated (UT) or treated with freshly dissolved 10 μM human IAPP (hIAPP) or mouse IAPP (mIAPP) for 24 hours. J, Rates of ER calcium-depleted INS1 832/13 cells untreated (UT) or treated with 1 mM dithiothreitol or 1 mM H 2 O 2 for 1 hour. K, Rates of ER calcium-depleted INS-1 GC cells untreated or treated with cytokines (100 ng/mL IL1-β, 100 ng/mL TNFα, and 100 ng/mL IFN-γ) for 24 hours. All values are means ± SEM (n = 3). *, P
    Figure Legend Snippet: β-Cell stressors induce ER calcium depletion. A, Rates of ER calcium-depleted INS-1 832/13 cells treated with indicated concentrations of glucose for 48 hours. B and C, Rates of ER calcium-depleted cells (B) and cell death (C) in INS-1 832/13 cells treated with 25 mM glucose (HG) for the indicated periods of time. D, Rates of ER calcium-depleted cells in INS1 832/13 treated with or without 25 mM 2-deoxyglucose (2DG) together with 25 mM high glucose (HG) for 48 hours. E, Rates of ER calcium-depleted INS-1 832/13 cells untreated or treated with 0.5 mM palmitic acid for the indicated periods of time. F, Rates of ER calcium-depleted INS-1 832/13 cells untreated (BSA) or treated with 0.5 mM oleic acid (OA), 0.5 mM palmitic acid (PA), 0.5 mM palmitic acid combined with high glucose (PA+HG), or 0.5 mM palmitic acid combined with 0.5 mM oleic acid (PA+OA) for 24 hours. G, Cytosolic calcium levels in INS1 832/13 cells untreated (UT) or treated with 0.5 mM palmitic acid (PA) or 0.5 mM palmitic acid together with high glucose (PA+HG) for 24 hours. The calcium efflux from the ER was induced by 3 μM thapsigargin at time 0 (arrow), and the calcium level was determined by Fluo-4 staining. H, Rates of cell death in INS-1 832/13 cells untreated (UT) or treated with 0.5 mM palmitic acid (PA), 0.5 mM oleic acid (OA), or both (PA+OA) were determined by propidium iodide staining. I, Rates of ER calcium-depleted INS-1 832/13 cells untreated (UT) or treated with freshly dissolved 10 μM human IAPP (hIAPP) or mouse IAPP (mIAPP) for 24 hours. J, Rates of ER calcium-depleted INS1 832/13 cells untreated (UT) or treated with 1 mM dithiothreitol or 1 mM H 2 O 2 for 1 hour. K, Rates of ER calcium-depleted INS-1 GC cells untreated or treated with cytokines (100 ng/mL IL1-β, 100 ng/mL TNFα, and 100 ng/mL IFN-γ) for 24 hours. All values are means ± SEM (n = 3). *, P

    Techniques Used: Staining

    17) Product Images from "Redox-responsive F127-folate/F127-disulfide bond-d-α-tocopheryl polyethylene glycol 1000 succinate/P123 mixed micelles loaded with paclitaxel for the reversal of multidrug resistance in tumors"

    Article Title: Redox-responsive F127-folate/F127-disulfide bond-d-α-tocopheryl polyethylene glycol 1000 succinate/P123 mixed micelles loaded with paclitaxel for the reversal of multidrug resistance in tumors

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S152395

    The particle size changes of ( A ) TPGS, ( B ) FSST, and ( C ) FT in different incubation times with different DTT concentrations (mean ± SD, n=3). Abbreviations: DTT, dithiothreitol; FSST, F127-disulfide bond-TPGS; FT, F127-TPGS; TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.
    Figure Legend Snippet: The particle size changes of ( A ) TPGS, ( B ) FSST, and ( C ) FT in different incubation times with different DTT concentrations (mean ± SD, n=3). Abbreviations: DTT, dithiothreitol; FSST, F127-disulfide bond-TPGS; FT, F127-TPGS; TPGS, d -α-tocopheryl polyethylene glycol 1000 succinate.

    Techniques Used: Incubation

    18) Product Images from "Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA"

    Article Title: Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S82413

    Agarose gel electrophoresis assay. Notes: ( A ) Agarose gel electrophoresis of pGL3 binding affinity for SHR and C-SHR-3 at various N/P ratios. ( B ) Heparin were used to evaluate the release of pGL3 from C-SHR-3/pGL3 complexes at an N/P ratio of 5. Abbreviations: C-SHR, disulfide cross-linked stearylated polyarginine peptide modified with histidine; SHR, non-cross-linked stearylated polyarginine peptide; DTT, dithiothreitol; pDNA, plasmid DNA.
    Figure Legend Snippet: Agarose gel electrophoresis assay. Notes: ( A ) Agarose gel electrophoresis of pGL3 binding affinity for SHR and C-SHR-3 at various N/P ratios. ( B ) Heparin were used to evaluate the release of pGL3 from C-SHR-3/pGL3 complexes at an N/P ratio of 5. Abbreviations: C-SHR, disulfide cross-linked stearylated polyarginine peptide modified with histidine; SHR, non-cross-linked stearylated polyarginine peptide; DTT, dithiothreitol; pDNA, plasmid DNA.

    Techniques Used: Agarose Gel Electrophoresis, Binding Assay, Modification, Plasmid Preparation

    19) Product Images from "Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody"

    Article Title: Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody

    Journal: Toxins

    doi: 10.3390/toxins8040100

    Sequential [ 32 P]-ADP-ribosylation of Rho. CHO cells ( a ) and HT22 cells ( c ) were treated with different C3 concentrations for indicated time points. Subsequently, cell lysates were incubated with 1 µM C3 and 1 µCi [ 32 P]-NAD in 20 µL of buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.3), 10 mM MgCl 2 , 10 mM dithiothreitol, 10 mM thymidine, and 10 µM nicotinamide adenine dinucleotide (NAD) at 37 °C for 30 min. The reaction was terminated by addition of Laemmli sample buffer and then incubated at 95 °C for 10 min. Samples were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the [ 32 P]-ADP-ribosylated Rho was analyzed by phosphorimaging. Representative phosphorimaging of [ 32 P]-ADP-ribosylated Rho and the Coomassie brilliant blue stained SDS-PAGE gels as loading control are presented ( n = 4). Molecular masses are indicated in kDa. M = Marker SDS7 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany); and ( b , d ) diagrams depict cellular level of unmodified Rho (● control, ■ 10 nM C3, ▲ 100 nM C3, ★ 1000 nM C3). Cellular level of unmodified Rho was determined by densitometrically quantification of intensity of sequential [ 32 P]-ADP-ribosylated Rho using Kodak software 1D, Version 3.5. The signal intensity of [ 32 P]-ADP-ribosylated Rho from untreated control cells were set as 1. Statistical differences were determined by two-sided Student’s t test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).
    Figure Legend Snippet: Sequential [ 32 P]-ADP-ribosylation of Rho. CHO cells ( a ) and HT22 cells ( c ) were treated with different C3 concentrations for indicated time points. Subsequently, cell lysates were incubated with 1 µM C3 and 1 µCi [ 32 P]-NAD in 20 µL of buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.3), 10 mM MgCl 2 , 10 mM dithiothreitol, 10 mM thymidine, and 10 µM nicotinamide adenine dinucleotide (NAD) at 37 °C for 30 min. The reaction was terminated by addition of Laemmli sample buffer and then incubated at 95 °C for 10 min. Samples were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the [ 32 P]-ADP-ribosylated Rho was analyzed by phosphorimaging. Representative phosphorimaging of [ 32 P]-ADP-ribosylated Rho and the Coomassie brilliant blue stained SDS-PAGE gels as loading control are presented ( n = 4). Molecular masses are indicated in kDa. M = Marker SDS7 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany); and ( b , d ) diagrams depict cellular level of unmodified Rho (● control, ■ 10 nM C3, ▲ 100 nM C3, ★ 1000 nM C3). Cellular level of unmodified Rho was determined by densitometrically quantification of intensity of sequential [ 32 P]-ADP-ribosylated Rho using Kodak software 1D, Version 3.5. The signal intensity of [ 32 P]-ADP-ribosylated Rho from untreated control cells were set as 1. Statistical differences were determined by two-sided Student’s t test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Marker, Software

    20) Product Images from "IRG1 and iNOS act redundantly with other interferon gamma-induced factors to restrict intracellular replication of Legionella pneumophila"

    Article Title: IRG1 and iNOS act redundantly with other interferon gamma-induced factors to restrict intracellular replication of Legionella pneumophila

    Journal: bioRxiv

    doi: 10.1101/731182

    Nos2 and Acod1 transcription segregates with conditions permissive and restrictive for L. pneumophila replication in BMMs. ( A and B ) Histograms displaying transcripts per million (TPM) of Nos2 , Acod1 , Irgm1 , Irgm3 , Cybb , and Casp11 measured by RNAseq recovered from 1.0 × 10 6 BMMs/condition stimulated for 18 hours with 100 ng/ml Pam3CSK4 (Pam) alone or in combination with 6.0 ng/ml IFNγ, IFNγ + 2.0 mM 2DG, IFNγ + 2.0 μM geldanamycin (Geld), IFNγ + 1.0 μg/ml brefeldin A (BfA), IFNγ + 2.0 mM dithiothreitol (DTT), IFNγ + 10.0 μM tunicamycin (Tunic), IFNγ + 25.0 nM thapsigargin (Thaps).
    Figure Legend Snippet: Nos2 and Acod1 transcription segregates with conditions permissive and restrictive for L. pneumophila replication in BMMs. ( A and B ) Histograms displaying transcripts per million (TPM) of Nos2 , Acod1 , Irgm1 , Irgm3 , Cybb , and Casp11 measured by RNAseq recovered from 1.0 × 10 6 BMMs/condition stimulated for 18 hours with 100 ng/ml Pam3CSK4 (Pam) alone or in combination with 6.0 ng/ml IFNγ, IFNγ + 2.0 mM 2DG, IFNγ + 2.0 μM geldanamycin (Geld), IFNγ + 1.0 μg/ml brefeldin A (BfA), IFNγ + 2.0 mM dithiothreitol (DTT), IFNγ + 10.0 μM tunicamycin (Tunic), IFNγ + 25.0 nM thapsigargin (Thaps).

    Techniques Used:

    Differential effect of UPR stress stimuli on rescue of L. pneumophila replication in IFNγ-stimulated macrophages. ( A ) RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected WT C57BL/6 BMMs stimulated for 48 hours post-infection with 6.0 ng/mL IFNγ, 2.0 mM 2DG, 2.0 μM geldanamycin (geld.), 1.0 μg/ml brefeldin A (BfA), and 2.0 mM dithiothreitol (DTT) as indicated. ( B ) RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected WT C57BL/6 BMMs stimulated for 48 hours post-infection with 6.0 ng/mL IFNγ, 10.0 μM tunicamycin (tunic.), and 25.0 nM thapsigargin (thaps.) as indicated. ( C ) RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected WT C57BL/6 BMMs stimulated for 48 hours post-infection with 6.0 ng/mL IFNγ, 2.0 mM 2DG, 2.0 μM geldanamycin, and 0.1 μM ISRIB as indicated. *** = p
    Figure Legend Snippet: Differential effect of UPR stress stimuli on rescue of L. pneumophila replication in IFNγ-stimulated macrophages. ( A ) RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected WT C57BL/6 BMMs stimulated for 48 hours post-infection with 6.0 ng/mL IFNγ, 2.0 mM 2DG, 2.0 μM geldanamycin (geld.), 1.0 μg/ml brefeldin A (BfA), and 2.0 mM dithiothreitol (DTT) as indicated. ( B ) RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected WT C57BL/6 BMMs stimulated for 48 hours post-infection with 6.0 ng/mL IFNγ, 10.0 μM tunicamycin (tunic.), and 25.0 nM thapsigargin (thaps.) as indicated. ( C ) RLU from LP02 Δ flaA Δ uhpC lux L. pneumophila from infected WT C57BL/6 BMMs stimulated for 48 hours post-infection with 6.0 ng/mL IFNγ, 2.0 mM 2DG, 2.0 μM geldanamycin, and 0.1 μM ISRIB as indicated. *** = p

    Techniques Used: Infection

    Validation of UPR gene expression by UPR stimuli and inhibition of ATF4-dependent gene expression by ISRIB. ( A ) Histograms displaying transcripts per million (TPM) of Hspa5 (heat shock protein 5, aka BIP), Trib3 (tribbles pseudokinase 3), Dnajb3 (DnaJ heat shock protein family (Hsp40) member B3), Pdia4 (protein disulfide isomerase associated 4), Manf (mesencephalic astrocyte-derived neurotrophic factor), and Hyou1 (hypoxia up-regulated 1) measured by RNAseq recovered from 1.0 × 10 6 BMMs/condition stimulated for 18 hours with 100 ng/ml Pam3CSK4 (Pam) alone or in combination with 6.0 ng/ml IFNγ, IFNγ + 2.0 mM 2DG, IFNγ + 2.0 μM geldanamycin (Geld), IFNγ + 1.0 μg/ml brefeldin A (BfA), IFNγ + 2.0 mM dithiothreitol (DTT), IFNγ + 10.0 μM tunicamycin (Tunic), or IFNγ + 25.0 nM thapsigargin (Thaps). These genes are a subset associated with gene ontology term GO:0034976, response to endoplasmic reticulum stress. ( B ) Histograms displaying TPM of Ddit3 (DNA-damage inducible transcript 3, aka CHOP), Atf3 (activating transcription factor 3), and Asns (asparagine synthetase) measured by RNAseq recovered from 1.0 × 10 6 BMMs/condition stimulated for 18 hours with 100 ng/ml Pam3CSK4 (Pam) alone or in combination with 6.0 ng/ml IFNγ, IFNγ + 2.0 mM 2DG, or IFNγ + 2.0 mM 2DG + 0.1 μM ISRIB. These genes are associated with PERK/ATF4-dependent gene expression ( 49 ).
    Figure Legend Snippet: Validation of UPR gene expression by UPR stimuli and inhibition of ATF4-dependent gene expression by ISRIB. ( A ) Histograms displaying transcripts per million (TPM) of Hspa5 (heat shock protein 5, aka BIP), Trib3 (tribbles pseudokinase 3), Dnajb3 (DnaJ heat shock protein family (Hsp40) member B3), Pdia4 (protein disulfide isomerase associated 4), Manf (mesencephalic astrocyte-derived neurotrophic factor), and Hyou1 (hypoxia up-regulated 1) measured by RNAseq recovered from 1.0 × 10 6 BMMs/condition stimulated for 18 hours with 100 ng/ml Pam3CSK4 (Pam) alone or in combination with 6.0 ng/ml IFNγ, IFNγ + 2.0 mM 2DG, IFNγ + 2.0 μM geldanamycin (Geld), IFNγ + 1.0 μg/ml brefeldin A (BfA), IFNγ + 2.0 mM dithiothreitol (DTT), IFNγ + 10.0 μM tunicamycin (Tunic), or IFNγ + 25.0 nM thapsigargin (Thaps). These genes are a subset associated with gene ontology term GO:0034976, response to endoplasmic reticulum stress. ( B ) Histograms displaying TPM of Ddit3 (DNA-damage inducible transcript 3, aka CHOP), Atf3 (activating transcription factor 3), and Asns (asparagine synthetase) measured by RNAseq recovered from 1.0 × 10 6 BMMs/condition stimulated for 18 hours with 100 ng/ml Pam3CSK4 (Pam) alone or in combination with 6.0 ng/ml IFNγ, IFNγ + 2.0 mM 2DG, or IFNγ + 2.0 mM 2DG + 0.1 μM ISRIB. These genes are associated with PERK/ATF4-dependent gene expression ( 49 ).

    Techniques Used: Expressing, Inhibition, Derivative Assay

    21) Product Images from "Kinetic Studies of Calcium-Induced Calcium Release in Cardiac Sarcoplasmic Reticulum Vesicles"

    Article Title: Kinetic Studies of Calcium-Induced Calcium Release in Cardiac Sarcoplasmic Reticulum Vesicles

    Journal: Biophysical Journal

    doi:

    Reversibility of the effects of thimerosal. Calcium release was induced at pCa 5, 1.2 mM free ATP, 0.8 mM free [Mg 2+ ]. ( A ) Native cardiac SR vesicles. ( B ) Vesicles incubated with 250 μ M thimerosal for 5 min. ( C ) Vesicles incubated with 250 μ M thimerosal for 5 min and then for 5 min with 5 mM dithiothreitol.
    Figure Legend Snippet: Reversibility of the effects of thimerosal. Calcium release was induced at pCa 5, 1.2 mM free ATP, 0.8 mM free [Mg 2+ ]. ( A ) Native cardiac SR vesicles. ( B ) Vesicles incubated with 250 μ M thimerosal for 5 min. ( C ) Vesicles incubated with 250 μ M thimerosal for 5 min and then for 5 min with 5 mM dithiothreitol.

    Techniques Used: Incubation

    22) Product Images from "In vitro Conversion of Vinyl to Formyl Groups in Naturally Occurring Chlorophylls"

    Article Title: In vitro Conversion of Vinyl to Formyl Groups in Naturally Occurring Chlorophylls

    Journal: Scientific Reports

    doi: 10.1038/srep06069

    Dithiothreitol and benzyl mercaptan also facilitate the conversion of the C3 vinyl of Chl a (a) and Chl b (b) to a formyl. RP-HPLC chromatograms (top panels) demonstrate retention times of the 3 1 -formyl derivatives of Chl a and Chl b were identical and are marked with vertical lines. Spectra (bottom panels) were identical to the 3 1 -formyl derivatives formed in the presence of β-mercaptoethanol (β-merc.). Note, in the case of the reaction of Chl a with benzyl mercaptan, another product spectrally similar to Chl a has a similar retention time to Chl d , causing the mixed online absorption spectrum. Q y for [3-formyl]-Chl a (Chl d ) and [3-formyl]-Chl b are marked. Dotted line, β-mercaptoethanol; dashed line, dithiothreitol; solid line, benzyl mercaptan.
    Figure Legend Snippet: Dithiothreitol and benzyl mercaptan also facilitate the conversion of the C3 vinyl of Chl a (a) and Chl b (b) to a formyl. RP-HPLC chromatograms (top panels) demonstrate retention times of the 3 1 -formyl derivatives of Chl a and Chl b were identical and are marked with vertical lines. Spectra (bottom panels) were identical to the 3 1 -formyl derivatives formed in the presence of β-mercaptoethanol (β-merc.). Note, in the case of the reaction of Chl a with benzyl mercaptan, another product spectrally similar to Chl a has a similar retention time to Chl d , causing the mixed online absorption spectrum. Q y for [3-formyl]-Chl a (Chl d ) and [3-formyl]-Chl b are marked. Dotted line, β-mercaptoethanol; dashed line, dithiothreitol; solid line, benzyl mercaptan.

    Techniques Used: High Performance Liquid Chromatography

    23) Product Images from "Biological functions of the disulfides in bovine pancreatic deoxyribonuclease"

    Article Title: Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

    Journal:

    doi: 10.1110/ps.03438204

    The thioredoxin-like activity of bpDNase. ( A ) The dithiothreitol-dependent reduction of insulin by thioredoxin or bpDNase. The reaction mixture contained, in a final volume of 0.6 mL: 0.1M potassium phosphate (pH 7.0), 2 mM EDTA, 0.13 mM bovine insulin,
    Figure Legend Snippet: The thioredoxin-like activity of bpDNase. ( A ) The dithiothreitol-dependent reduction of insulin by thioredoxin or bpDNase. The reaction mixture contained, in a final volume of 0.6 mL: 0.1M potassium phosphate (pH 7.0), 2 mM EDTA, 0.13 mM bovine insulin,

    Techniques Used: Activity Assay

    24) Product Images from "Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase"

    Article Title: Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase

    Journal: Scientific Reports

    doi: 10.1038/srep06574

    CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.
    Figure Legend Snippet: CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.

    Techniques Used:

    25) Product Images from "Improved measurements of scant hydrogen peroxide enable experiments that define its threshold of toxicity for Escherichia coli"

    Article Title: Improved measurements of scant hydrogen peroxide enable experiments that define its threshold of toxicity for Escherichia coli

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2018.03.025

    Thiol species interfere with H 2 O 2 detection, but adjustments can correct for the problem (A) Dithiothreitol suppresses half the fluorescence otherwise produced in an HRP/AR/H 2 O 2 reaction containing 0.5 μM H 2 O 2 . (B) Even at high levels of dithiothreitol (here, 100 μM), standard curves permit H 2 O 2 quantification. (C) Thiol species only slowly scavenge H 2 O 2 . Dithiothreitol (50 μM) and 50 μM H 2 O 2 were coincubated in RT KPi/DTPA buffer at the indicated pH values, and H 2 O 2 was periodically quantified. Data indicate a rate constant for H 2 O 2 reduction by the DTT thiolate anion of 15 M −1 s −1 , which equates to an H 2 O 2 half-life of 3 hours in 100 μM DTT.
    Figure Legend Snippet: Thiol species interfere with H 2 O 2 detection, but adjustments can correct for the problem (A) Dithiothreitol suppresses half the fluorescence otherwise produced in an HRP/AR/H 2 O 2 reaction containing 0.5 μM H 2 O 2 . (B) Even at high levels of dithiothreitol (here, 100 μM), standard curves permit H 2 O 2 quantification. (C) Thiol species only slowly scavenge H 2 O 2 . Dithiothreitol (50 μM) and 50 μM H 2 O 2 were coincubated in RT KPi/DTPA buffer at the indicated pH values, and H 2 O 2 was periodically quantified. Data indicate a rate constant for H 2 O 2 reduction by the DTT thiolate anion of 15 M −1 s −1 , which equates to an H 2 O 2 half-life of 3 hours in 100 μM DTT.

    Techniques Used: Fluorescence, Produced

    26) Product Images from "Mon1 Is Essential for Fungal Virulence and Stress Survival in Cryptococcus neoformans"

    Article Title: Mon1 Is Essential for Fungal Virulence and Stress Survival in Cryptococcus neoformans

    Journal: Mycobiology

    doi: 10.1080/12298093.2018.1468053

    Phenotypes of the Cn mon1 Δ mutant following exposure to various stresses. Spot dilution assays with WT (H99), Cn cna1 Δ (KK1), Cn mon1 Δ (HP55 and HP56), and Cn mon1 + Cn MON1 (HPC3 and HPC4). Cells were incubated overnight, diluted 10-fold, and plated on YPD agar. Plated cells were incubated for 2 days at 30, 37, 38, and 39 °C. (A) Serially diluted cells were also plated on YPD agar without or with (B) dithiothreitol (DTT), (C) sodium dodecylsulfate (SDS), (D) calcofluor white, (E) congo red, and (F) fluconazole at the indicated concentrations. Results shown are representative of two independent experiments.
    Figure Legend Snippet: Phenotypes of the Cn mon1 Δ mutant following exposure to various stresses. Spot dilution assays with WT (H99), Cn cna1 Δ (KK1), Cn mon1 Δ (HP55 and HP56), and Cn mon1 + Cn MON1 (HPC3 and HPC4). Cells were incubated overnight, diluted 10-fold, and plated on YPD agar. Plated cells were incubated for 2 days at 30, 37, 38, and 39 °C. (A) Serially diluted cells were also plated on YPD agar without or with (B) dithiothreitol (DTT), (C) sodium dodecylsulfate (SDS), (D) calcofluor white, (E) congo red, and (F) fluconazole at the indicated concentrations. Results shown are representative of two independent experiments.

    Techniques Used: Mutagenesis, Incubation

    27) Product Images from "Effects of Reactive Oxygen and Nitrogen Metabolites on RANTES- and IL-5-Induced Eosinophil Chemotactic Activity in Vitro"

    Article Title: Effects of Reactive Oxygen and Nitrogen Metabolites on RANTES- and IL-5-Induced Eosinophil Chemotactic Activity in Vitro

    Journal: The American Journal of Pathology

    doi:

    Effect of dithiothreitol and deferoxamine on peroxynitrite-induced attenuation of ECA by RANTES ( A ) or IL-5 ( B ). RANTES or IL-5 were incubated with peroxynitrite for 2 hours in the presence of dithiothreitol and deferoxamine at the concentration indicated; n = 4 each condition. ECA is on the ordinate and the experimental groups are on the abscissa. * P
    Figure Legend Snippet: Effect of dithiothreitol and deferoxamine on peroxynitrite-induced attenuation of ECA by RANTES ( A ) or IL-5 ( B ). RANTES or IL-5 were incubated with peroxynitrite for 2 hours in the presence of dithiothreitol and deferoxamine at the concentration indicated; n = 4 each condition. ECA is on the ordinate and the experimental groups are on the abscissa. * P

    Techniques Used: Incubation, Concentration Assay

    28) Product Images from "Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2"

    Article Title: Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-13-0588

    Reversible S-nitrosylation of TSC2 blocks TSC1-TSC2 dimerization and upregulates Rheb activity A375 cells were cultured with the indicated concentrations of L-NIL, the NO donor S-nitroso-acetyl-penicillamine (SNAP), or the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (PTIO) for 16 Hours prior to harvest and preparation of protein lysates. As a negative control, parallel lysates were exposed to 10 mM of SH-reducing agent dithiothreitol (DTT) for 1 hour in vitro . ( A ) Total S-nitrosylated protein levels were determined by biotin switch assay and anti-biotin immunoblot; “unbiotinylated” shows endogenous biotin levels. ( B ) S-Nitrosylation of TSC2 in biotin switch assay followed by immunoprecipitation with streptavidine agarose and immunoblot analysis with anti-TSC2 polyclonal antibody. “SNO” (S-nitrosothiol) indicates biotinylated (nitrosylated) TSC2 levels following biotin switch. “Total” indicates total TSC2 levels prior to immunoprecipitation. Actin was used as a loading control. ( C ) Effect of iNOS/NO on TSC2/TSC1 dimerization. A375 cells were treated with NO donors DEANONOate or SNAP, or the selective iNOS inhibitor L-NIL, and cell lysates were immunoprecipitated (IP) with anti-TSC2 antibody. Immunocomplexes (upper panel) and crude lysates (lower panel) were immunoblotted with anti-TSC1, -TSC2, or –actin antibody. The ratio of TSC1 to TSC2 was calculated and normalized against the amount of TSC2 present after the immnunoprecipitation ( D ) Effect of iNOS/NO on Rheb activation. A375 cells were transfected with iNOS-targeting siRNA or scrambled control and cultured for 48 hours. Western blots for activated Rheb (Rheb/GTP) total Rheb, including the inactive GDP-Bound form (GDP), and actin loading control were performed. Actived Rheb was quantified by densitometry and normalized to total Rheb levels. All graphs represent data from 3 independent experiments.
    Figure Legend Snippet: Reversible S-nitrosylation of TSC2 blocks TSC1-TSC2 dimerization and upregulates Rheb activity A375 cells were cultured with the indicated concentrations of L-NIL, the NO donor S-nitroso-acetyl-penicillamine (SNAP), or the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (PTIO) for 16 Hours prior to harvest and preparation of protein lysates. As a negative control, parallel lysates were exposed to 10 mM of SH-reducing agent dithiothreitol (DTT) for 1 hour in vitro . ( A ) Total S-nitrosylated protein levels were determined by biotin switch assay and anti-biotin immunoblot; “unbiotinylated” shows endogenous biotin levels. ( B ) S-Nitrosylation of TSC2 in biotin switch assay followed by immunoprecipitation with streptavidine agarose and immunoblot analysis with anti-TSC2 polyclonal antibody. “SNO” (S-nitrosothiol) indicates biotinylated (nitrosylated) TSC2 levels following biotin switch. “Total” indicates total TSC2 levels prior to immunoprecipitation. Actin was used as a loading control. ( C ) Effect of iNOS/NO on TSC2/TSC1 dimerization. A375 cells were treated with NO donors DEANONOate or SNAP, or the selective iNOS inhibitor L-NIL, and cell lysates were immunoprecipitated (IP) with anti-TSC2 antibody. Immunocomplexes (upper panel) and crude lysates (lower panel) were immunoblotted with anti-TSC1, -TSC2, or –actin antibody. The ratio of TSC1 to TSC2 was calculated and normalized against the amount of TSC2 present after the immnunoprecipitation ( D ) Effect of iNOS/NO on Rheb activation. A375 cells were transfected with iNOS-targeting siRNA or scrambled control and cultured for 48 hours. Western blots for activated Rheb (Rheb/GTP) total Rheb, including the inactive GDP-Bound form (GDP), and actin loading control were performed. Actived Rheb was quantified by densitometry and normalized to total Rheb levels. All graphs represent data from 3 independent experiments.

    Techniques Used: Activity Assay, Cell Culture, Negative Control, In Vitro, Biotin Switch Assay, Immunoprecipitation, Activation Assay, Transfection, Western Blot

    29) Product Images from "Differentiation renders susceptibility to excitotoxicity in HT22 neurons ☆"

    Article Title: Differentiation renders susceptibility to excitotoxicity in HT22 neurons ☆

    Journal: Neural Regeneration Research

    doi: 10.3969/j.issn.1673-5374.2013.14.006

    Morphological analysis on the effect of N-methyl-D-aspartate (NMDA) receptor antagonists and antioxidants on glutamate toxicity in undifferentiated and differentiated HT22 cells. Propidium iodide (red) and Hoechst 33342 (blue) staining were used as apoptosis markers on undifferentiated and differentiated HT22 cells following exposure to glutamate (Glu) half-effective concentrations (1.8 mmol/L and 50 µmol/L, respectively) and interfering drugs (MK-801 at 20 µmol/L, memantine (Mem) at 12 µmol/L and dithiothreitol (DTT) at 250 µmol/L). Apoptotic cells were stained with propidium iodide. Staining revealed that glutamate-induced cytotoxicity on undifferentiated HT22 cells could be prevented by dithiothreitol (an antioxidant), while dithiothreitol exhibited little effect on differentiated cells. Meanwhile, MK-801 and memantine (as NMDA receptor antagonists) could protect differentiated cells from glutamate toxicity, but had no effect on undifferentiated HT22 cells.
    Figure Legend Snippet: Morphological analysis on the effect of N-methyl-D-aspartate (NMDA) receptor antagonists and antioxidants on glutamate toxicity in undifferentiated and differentiated HT22 cells. Propidium iodide (red) and Hoechst 33342 (blue) staining were used as apoptosis markers on undifferentiated and differentiated HT22 cells following exposure to glutamate (Glu) half-effective concentrations (1.8 mmol/L and 50 µmol/L, respectively) and interfering drugs (MK-801 at 20 µmol/L, memantine (Mem) at 12 µmol/L and dithiothreitol (DTT) at 250 µmol/L). Apoptotic cells were stained with propidium iodide. Staining revealed that glutamate-induced cytotoxicity on undifferentiated HT22 cells could be prevented by dithiothreitol (an antioxidant), while dithiothreitol exhibited little effect on differentiated cells. Meanwhile, MK-801 and memantine (as NMDA receptor antagonists) could protect differentiated cells from glutamate toxicity, but had no effect on undifferentiated HT22 cells.

    Techniques Used: Staining

    Effect of antioxidant on glutamate toxicity in differentiated and undifferentiated HT22 cells. Dithiothreitol (DTT; 250 µmol/L) was added as an antioxidant to reduce cytotoxicity in HT22 cells by oxidative stress. 1.8 mmol/L and 50 µmol/L glutamate (Glu) was added to undifferentiated and differentiated HT22 cells separately to induce basic cytotoxicity. Cell toxicity and viability were estimated using the lactate dehydrogenase (A) and methyl thiazolyl tetrazolium (MTT; B) assays, respectively. Sterile PBS is marked by ‘cont’. All quantitative data were expressed as mean ± SEM, and analyzed using appropriate analysis of variance, followed by post hoc comparison of the means using Fisher least significant difference test and Bonferroni correction. After DTT was added, undifferentiated HT22 cells were rescued from oxidative stress as determined by both the lactate dehydrogenase (cytotoxicity from 45.57% to 17.23%; a P
    Figure Legend Snippet: Effect of antioxidant on glutamate toxicity in differentiated and undifferentiated HT22 cells. Dithiothreitol (DTT; 250 µmol/L) was added as an antioxidant to reduce cytotoxicity in HT22 cells by oxidative stress. 1.8 mmol/L and 50 µmol/L glutamate (Glu) was added to undifferentiated and differentiated HT22 cells separately to induce basic cytotoxicity. Cell toxicity and viability were estimated using the lactate dehydrogenase (A) and methyl thiazolyl tetrazolium (MTT; B) assays, respectively. Sterile PBS is marked by ‘cont’. All quantitative data were expressed as mean ± SEM, and analyzed using appropriate analysis of variance, followed by post hoc comparison of the means using Fisher least significant difference test and Bonferroni correction. After DTT was added, undifferentiated HT22 cells were rescued from oxidative stress as determined by both the lactate dehydrogenase (cytotoxicity from 45.57% to 17.23%; a P

    Techniques Used: MTT Assay

    30) Product Images from "Hepcidin induction by transgenic overexpression of Hfe does not require the Hfe cytoplasmic tail, but does require hemojuvelin"

    Article Title: Hepcidin induction by transgenic overexpression of Hfe does not require the Hfe cytoplasmic tail, but does require hemojuvelin

    Journal: Blood

    doi: 10.1182/blood-2010-04-277954

    Hfe transgenes do not induce endoplasmic reticulum (ER) stress or the unfolded protein response (UPR) . Dithiothreitol (DTT) induction (A-B) of the endoplasmic reticulum (ER) stress response in mouse embryonic fibroblast (MEF) cells. (A) Semiquantitative
    Figure Legend Snippet: Hfe transgenes do not induce endoplasmic reticulum (ER) stress or the unfolded protein response (UPR) . Dithiothreitol (DTT) induction (A-B) of the endoplasmic reticulum (ER) stress response in mouse embryonic fibroblast (MEF) cells. (A) Semiquantitative

    Techniques Used:

    31) Product Images from "Nitro-Oleic Acid Inhibits Firing and Activates TRPV1- and TRPA1-Mediated Inward Currents in Dorsal Root Ganglion Neurons from Adult Male Rats"

    Article Title: Nitro-Oleic Acid Inhibits Firing and Activates TRPV1- and TRPA1-Mediated Inward Currents in Dorsal Root Ganglion Neurons from Adult Male Rats

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.109.163154

    Block of OA-NO 2 -evoked currents by TRP antagonists. A, inward currents induced by increasing concentrations of OA-NO 2 . Block of the OA-NO 2 -evoked current by a TRPA1 antagonist (HC-030031; 5 μM). B, block of an OA-NO 2 -evoked current by the reducing agent dithiothreitol (10 mM). C, partial block of an OA-NO 2 -evoked current by the TRPV1 antagonist diarylpiperazine (5 μM), and the remaining current was blocked by subsequent addition of HC-030031 (5 μM). D, average block of an OA-NO 2 -evoked current by each antagonist alone or the combination of the two antagonists or by dithiothreitol. Inset shows average time course for prolonged application of OA-NO 2 (0.05 μM) in five neurons (empty circles) that was fitted with an equation consisting of one exponential for the rising phase and two exponentials for the decaying phase as explained under Materials and Methods . Inset shows the effect of diarylpiperazine (5 μM) in two neurons. Currents were normalized to the peak inward current in all cells. Statistical comparisons in D (**, p
    Figure Legend Snippet: Block of OA-NO 2 -evoked currents by TRP antagonists. A, inward currents induced by increasing concentrations of OA-NO 2 . Block of the OA-NO 2 -evoked current by a TRPA1 antagonist (HC-030031; 5 μM). B, block of an OA-NO 2 -evoked current by the reducing agent dithiothreitol (10 mM). C, partial block of an OA-NO 2 -evoked current by the TRPV1 antagonist diarylpiperazine (5 μM), and the remaining current was blocked by subsequent addition of HC-030031 (5 μM). D, average block of an OA-NO 2 -evoked current by each antagonist alone or the combination of the two antagonists or by dithiothreitol. Inset shows average time course for prolonged application of OA-NO 2 (0.05 μM) in five neurons (empty circles) that was fitted with an equation consisting of one exponential for the rising phase and two exponentials for the decaying phase as explained under Materials and Methods . Inset shows the effect of diarylpiperazine (5 μM) in two neurons. Currents were normalized to the peak inward current in all cells. Statistical comparisons in D (**, p

    Techniques Used: Blocking Assay

    32) Product Images from "Age-Dependent, Subunit Specific Action of Hydrogen Sulfide on GluN1/2A and GluN1/2B NMDA Receptors"

    Article Title: Age-Dependent, Subunit Specific Action of Hydrogen Sulfide on GluN1/2A and GluN1/2B NMDA Receptors

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00375

    The role of disulfide bonds reduction in the effects of NaHS on GluN1/2A and GluN1/2B receptors. Representative current traces activated by 100 μM NMDA + 30 μM glycine (5 s, black bar) in control, after application of 2 mM dithiothreitol (DTT) and 2 mM DTT+100 μM NaHS in HEK293T cells expressing GluN1/2A (A) or GluN1/2B (B) receptors. Statistical plot of NMDA induced total charge transfer in the presence of 2 mM DTT and DTT+NaHS. Each pair of connected circles corresponds to an individual HEK293T cell expressing GluN1/2A ( C ; n = 8) and GluN1/2B ( D ; n = 8). Boxes indicate 25–75 percentiles in control (white) and in NaHS (gray), black line—median, the circle inside—mean value, whiskers—minimal and maximal values, * p
    Figure Legend Snippet: The role of disulfide bonds reduction in the effects of NaHS on GluN1/2A and GluN1/2B receptors. Representative current traces activated by 100 μM NMDA + 30 μM glycine (5 s, black bar) in control, after application of 2 mM dithiothreitol (DTT) and 2 mM DTT+100 μM NaHS in HEK293T cells expressing GluN1/2A (A) or GluN1/2B (B) receptors. Statistical plot of NMDA induced total charge transfer in the presence of 2 mM DTT and DTT+NaHS. Each pair of connected circles corresponds to an individual HEK293T cell expressing GluN1/2A ( C ; n = 8) and GluN1/2B ( D ; n = 8). Boxes indicate 25–75 percentiles in control (white) and in NaHS (gray), black line—median, the circle inside—mean value, whiskers—minimal and maximal values, * p

    Techniques Used: Expressing

    33) Product Images from "Induction of the Unfolded Protein Response Drives Enhanced Metabolism and Chemoresistance in Glioma Cells"

    Article Title: Induction of the Unfolded Protein Response Drives Enhanced Metabolism and Chemoresistance in Glioma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073267

    Human glioma cells respond to persistent ER stress with UPR induction and rapid recovery of protein synthesis. Human glioma tissue culture models examined include: U87MG, U87+EGFR, and U87+EGFRvIII, from U87 parent lines stably transfected with a constitutively-active oncogenic EGFR and the extracellulary truncated EGFR variant III. ( A ) Northern blot analysis of human glioma tissue culture cells after a 4-hour treatment with the following pharmacological inducers of the UPR: 1mM dithiothreitol (DTT), 0.5 µM thapsigarin (Tg), or 2.5µg/ml tunicamycin(TM). Blots were probed for message levels of CHOP, XBP-1, GRP78/BiP, and GAPDH. ( B ) Levels of total newly-synthesized protein from 0–4 hours after a 1mM DTT treatment were assayed as TCA precipitable [ 35 S]-methionine-labeled protein. Protein synthesis rapidly declined and then rapidly recovered despite the presence of reducing agent in the culture. ( C ) The time course of eIF2α phosphorylation (“phospho- eIF2α”) was followed by immunoblotting during DTT treatment of U87MG cells, as was induction of spliced XBP-1 (“XBP-1(s)”. The actin loading control blot is a replicate blot. Following a 0-6 hour 1mM DTT treatment in U87 cell culture, ( D ) RNA was analyzed by Northern blot in a kinetic analysis of CHOP and XBP-1 mRNA. ( E ) Glioma cell cultures were labeled with [ 35 S]-methionine during a 0-4 hour 1mM DTT treatment. GRP94 was immunoprecipitated to detect newly-synthesized protein.
    Figure Legend Snippet: Human glioma cells respond to persistent ER stress with UPR induction and rapid recovery of protein synthesis. Human glioma tissue culture models examined include: U87MG, U87+EGFR, and U87+EGFRvIII, from U87 parent lines stably transfected with a constitutively-active oncogenic EGFR and the extracellulary truncated EGFR variant III. ( A ) Northern blot analysis of human glioma tissue culture cells after a 4-hour treatment with the following pharmacological inducers of the UPR: 1mM dithiothreitol (DTT), 0.5 µM thapsigarin (Tg), or 2.5µg/ml tunicamycin(TM). Blots were probed for message levels of CHOP, XBP-1, GRP78/BiP, and GAPDH. ( B ) Levels of total newly-synthesized protein from 0–4 hours after a 1mM DTT treatment were assayed as TCA precipitable [ 35 S]-methionine-labeled protein. Protein synthesis rapidly declined and then rapidly recovered despite the presence of reducing agent in the culture. ( C ) The time course of eIF2α phosphorylation (“phospho- eIF2α”) was followed by immunoblotting during DTT treatment of U87MG cells, as was induction of spliced XBP-1 (“XBP-1(s)”. The actin loading control blot is a replicate blot. Following a 0-6 hour 1mM DTT treatment in U87 cell culture, ( D ) RNA was analyzed by Northern blot in a kinetic analysis of CHOP and XBP-1 mRNA. ( E ) Glioma cell cultures were labeled with [ 35 S]-methionine during a 0-4 hour 1mM DTT treatment. GRP94 was immunoprecipitated to detect newly-synthesized protein.

    Techniques Used: Stable Transfection, Transfection, Variant Assay, Northern Blot, Synthesized, Labeling, Cell Culture, Immunoprecipitation

    34) Product Images from "Neutrophil-generated HOCl leads to non-specific thiol oxidation in phagocytized bacteria"

    Article Title: Neutrophil-generated HOCl leads to non-specific thiol oxidation in phagocytized bacteria

    Journal: eLife

    doi: 10.7554/eLife.32288

    Probe oxidation occurs within seconds after phagocytosis. ( A ) Quantitative fluorescence microscopy of the redox state of roGFP2-Orp1 in E. coli during phagocytosis. Stills of a movie observing an individual E. coli cell expressing roGFP2-Orp1 (indicated by an arrow) being attacked by a neutrophil-like PLB-985 cell. The neutrophil has already phagocytized another E. coli cell. Upon phagocytosis, the oxidation state changes within seconds (inset 65–80 s) as illustrated based on the false color scale indicated. See also Video 1 . ( B ) Control: E. coli cells in the absence of neutrophil-like cells untreated and treated with the reducing agent dithiothreitol (DTT) and the oxidizing agent Aldrithiol-2 (AT-2).
    Figure Legend Snippet: Probe oxidation occurs within seconds after phagocytosis. ( A ) Quantitative fluorescence microscopy of the redox state of roGFP2-Orp1 in E. coli during phagocytosis. Stills of a movie observing an individual E. coli cell expressing roGFP2-Orp1 (indicated by an arrow) being attacked by a neutrophil-like PLB-985 cell. The neutrophil has already phagocytized another E. coli cell. Upon phagocytosis, the oxidation state changes within seconds (inset 65–80 s) as illustrated based on the false color scale indicated. See also Video 1 . ( B ) Control: E. coli cells in the absence of neutrophil-like cells untreated and treated with the reducing agent dithiothreitol (DTT) and the oxidizing agent Aldrithiol-2 (AT-2).

    Techniques Used: Fluorescence, Microscopy, Expressing

    35) Product Images from "ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response"

    Article Title: ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response

    Journal:

    doi: 10.1182/blood-2007-09-112870

    Unfolded protein response up-regulates VEGF mRNA. HUVECs were treated for 6 hours in medium 199/2% FCS containing (A) 130 μM OxPAPC, 1 mM dithiothreitol (DTT), 3 μg/mL tunicamycin, 1 μg/mL thapsigargin, or 6 μg/mL brefeldin
    Figure Legend Snippet: Unfolded protein response up-regulates VEGF mRNA. HUVECs were treated for 6 hours in medium 199/2% FCS containing (A) 130 μM OxPAPC, 1 mM dithiothreitol (DTT), 3 μg/mL tunicamycin, 1 μg/mL thapsigargin, or 6 μg/mL brefeldin

    Techniques Used:

    36) Product Images from "Inhibition of TOR in Chlamydomonas reinhardtii Leads to Rapid Cysteine Oxidation Reflecting Sustained Physiological Changes"

    Article Title: Inhibition of TOR in Chlamydomonas reinhardtii Leads to Rapid Cysteine Oxidation Reflecting Sustained Physiological Changes

    Journal: Cells

    doi: 10.3390/cells8101171

    Workflow for proteomic oxidative cysteine analysis of C. reinhardtii with AZD8055 treatment. After protein extraction, reduced cysteine thiols are blocked with N -ethylmalemide (NEM), before reversibly oxidized cysteines are reduced using dithiothreitol (DTT). An oxidized cysteine resin-assisted capture method (OxRAC) is used to enrich proteins containing oxidized cysteines and samples are processed for bottom-up liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis.
    Figure Legend Snippet: Workflow for proteomic oxidative cysteine analysis of C. reinhardtii with AZD8055 treatment. After protein extraction, reduced cysteine thiols are blocked with N -ethylmalemide (NEM), before reversibly oxidized cysteines are reduced using dithiothreitol (DTT). An oxidized cysteine resin-assisted capture method (OxRAC) is used to enrich proteins containing oxidized cysteines and samples are processed for bottom-up liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis.

    Techniques Used: Protein Extraction, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    37) Product Images from "Propolis Modifies Collagen Types I and III Accumulation in the Matrix of Burnt Tissue"

    Article Title: Propolis Modifies Collagen Types I and III Accumulation in the Matrix of Burnt Tissue

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2013/423809

    (a) Typical electropherogram of α (I) subunits, oligomers β / γ (I), and collagen type I degradation products, released from healthy and burnt skin. Collagenous components released from tissue samples using pepsin were submitted to 4–15% gradient SDS-PAGE, in nonreducing conditions. (b) Interferences of collagen type III, in electrophoretic profiles of collagen type I components extracted from healthy and burned skin. Collagen components were submitted to electrophoresis in the absence of dithiothreitol (reducing disulfide bonds) and subsequently—after electrotransfer to Immobilon—subjected to reaction with collagen type III antibodies. Lane 1: components of collagen type I isolated from healthy skin. Lane 2: components of collagen type I isolated from burned skin treated with propolis. Lane 3: components of collagen type I isolated from burned skin treated with AgSD. Lane 4: components of collagen type I isolated from burned skin treated with propolis vehicle. Lane 5: components of collagen type I isolated from burned skin treated with NaCl.
    Figure Legend Snippet: (a) Typical electropherogram of α (I) subunits, oligomers β / γ (I), and collagen type I degradation products, released from healthy and burnt skin. Collagenous components released from tissue samples using pepsin were submitted to 4–15% gradient SDS-PAGE, in nonreducing conditions. (b) Interferences of collagen type III, in electrophoretic profiles of collagen type I components extracted from healthy and burned skin. Collagen components were submitted to electrophoresis in the absence of dithiothreitol (reducing disulfide bonds) and subsequently—after electrotransfer to Immobilon—subjected to reaction with collagen type III antibodies. Lane 1: components of collagen type I isolated from healthy skin. Lane 2: components of collagen type I isolated from burned skin treated with propolis. Lane 3: components of collagen type I isolated from burned skin treated with AgSD. Lane 4: components of collagen type I isolated from burned skin treated with propolis vehicle. Lane 5: components of collagen type I isolated from burned skin treated with NaCl.

    Techniques Used: SDS Page, Electrophoresis, Electrotransfer, Isolation

    38) Product Images from "Essential Roles of the Kar2/BiP Molecular Chaperone Downstream of the UPR Pathway in Cryptococcus neoformans"

    Article Title: Essential Roles of the Kar2/BiP Molecular Chaperone Downstream of the UPR Pathway in Cryptococcus neoformans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058956

    Kar2 is involved in ER stress response, thermotolerance, and maintenance of cell wall integrity downstream of the UPR pathway. The KAR2 overexpression strains [P H3 :KAR2 (YSB1751), ire1 Δ P H3 :KAR2 (YSB1741), and hxl1 Δ P H3 :KAR2 (YSB1745)] and their parent strains (WT H99 strain and ire1 Δ and hxl1 Δ mutants) were grown for 16 h at 30 o C in a liquid YPD medium, 10-fold serially diluted, and spotted on a YPD agar medium containing the indicated concentrations of tunicamycin (TM; A), dithiothreitol (DTT; B), diamide (C), calcofluor white (CFW; E), and congo-red (CR; E). Strains were incubated at 30°C or at 35, 37, or 39°C for thermotolerance test (D) for 3–4 days and were photographed.
    Figure Legend Snippet: Kar2 is involved in ER stress response, thermotolerance, and maintenance of cell wall integrity downstream of the UPR pathway. The KAR2 overexpression strains [P H3 :KAR2 (YSB1751), ire1 Δ P H3 :KAR2 (YSB1741), and hxl1 Δ P H3 :KAR2 (YSB1745)] and their parent strains (WT H99 strain and ire1 Δ and hxl1 Δ mutants) were grown for 16 h at 30 o C in a liquid YPD medium, 10-fold serially diluted, and spotted on a YPD agar medium containing the indicated concentrations of tunicamycin (TM; A), dithiothreitol (DTT; B), diamide (C), calcofluor white (CFW; E), and congo-red (CR; E). Strains were incubated at 30°C or at 35, 37, or 39°C for thermotolerance test (D) for 3–4 days and were photographed.

    Techniques Used: Over Expression, Incubation

    39) Product Images from "Unsaturated Fatty Acid-Induced Conformational Transitions and Aggregation of the Repeat Domain of Tau"

    Article Title: Unsaturated Fatty Acid-Induced Conformational Transitions and Aggregation of the Repeat Domain of Tau

    Journal: Molecules

    doi: 10.3390/molecules25112716

    Thioflavin T (ThT) fluorescence assays. ThT fluorescence intensity was monitored over time on samples containing: ( A ) 10 μM Tau 4RD and 30 μM fatty acid (FA); ( B ) 10 μM Tau 4RD and 300 μM FA; ( C ) 100 μM Tau 4RD and 300 μM FA. Protein and FAs were dissolved in phosphate buffer, pH 6.8, also containing excess dithiothreitol (DTT). Samples were incubated at 30 °C under intermittent agitation. Control measurements were performed on protein ( C ) and FAs ( B ). Aggregation kinetic curves for Tau 4RD in the presence of heparin (HEP) are displayed for comparison (grey circles). The displayed data are the mean ± SD of measurements performed in quadruplicate (no error bars are shown for control samples).
    Figure Legend Snippet: Thioflavin T (ThT) fluorescence assays. ThT fluorescence intensity was monitored over time on samples containing: ( A ) 10 μM Tau 4RD and 30 μM fatty acid (FA); ( B ) 10 μM Tau 4RD and 300 μM FA; ( C ) 100 μM Tau 4RD and 300 μM FA. Protein and FAs were dissolved in phosphate buffer, pH 6.8, also containing excess dithiothreitol (DTT). Samples were incubated at 30 °C under intermittent agitation. Control measurements were performed on protein ( C ) and FAs ( B ). Aggregation kinetic curves for Tau 4RD in the presence of heparin (HEP) are displayed for comparison (grey circles). The displayed data are the mean ± SD of measurements performed in quadruplicate (no error bars are shown for control samples).

    Techniques Used: Fluorescence, Incubation

    40) Product Images from "Possible involvement of membrane lipids peroxidation and oxidation of catalytically essential thiols of the cerebral transmembrane sodium pump as component mechanisms of iron-mediated oxidative stress-linked dysfunction of the pump's activity"

    Article Title: Possible involvement of membrane lipids peroxidation and oxidation of catalytically essential thiols of the cerebral transmembrane sodium pump as component mechanisms of iron-mediated oxidative stress-linked dysfunction of the pump's activity

    Journal: Redox Biology

    doi: 10.1016/j.redox.2014.12.015

    Effects of iron on the rate of dithiothreitol oxidation. The rate of oxidation was evaluated at the indicated times and concentrations of iron. Data are the means of five to seven independent experiments carried out in different days. Data are expressed as mean±SEM and post-hoc comparisons were done by Duncan's multiple range test. *Significant difference in relation to the control.
    Figure Legend Snippet: Effects of iron on the rate of dithiothreitol oxidation. The rate of oxidation was evaluated at the indicated times and concentrations of iron. Data are the means of five to seven independent experiments carried out in different days. Data are expressed as mean±SEM and post-hoc comparisons were done by Duncan's multiple range test. *Significant difference in relation to the control.

    Techniques Used:

    Related Articles

    In Vitro:

    Article Title: Characterization of pertussis-like toxin from Salmonella spp. that catalyzes ADP-ribosylation of G proteins
    Article Snippet: .. The in vitro ADP-ribosyltransferase reaction mixture (20 μl) contained 0.1 M Tris-HCl (pH 7.6), 0.1 mM ATP, 20 mM DTT, 5 mM thymidine, 10 μM β-NAD, and 0.1 μg of G protein from the bovine brain (Calbiochem-Novabiochem) or 5.6 μg of membrane proteins isolated from RAW 264.7 cells, and either the test substance or Ptx (Biomol, Hamburg, Germany). .. Ptx was preactivated by incubation in 50 mM Tris-HCl (pH 7.5) containing 50 mM DTT at 37 °C for 20 min.

    Isolation:

    Article Title: Characterization of pertussis-like toxin from Salmonella spp. that catalyzes ADP-ribosylation of G proteins
    Article Snippet: .. The in vitro ADP-ribosyltransferase reaction mixture (20 μl) contained 0.1 M Tris-HCl (pH 7.6), 0.1 mM ATP, 20 mM DTT, 5 mM thymidine, 10 μM β-NAD, and 0.1 μg of G protein from the bovine brain (Calbiochem-Novabiochem) or 5.6 μg of membrane proteins isolated from RAW 264.7 cells, and either the test substance or Ptx (Biomol, Hamburg, Germany). .. Ptx was preactivated by incubation in 50 mM Tris-HCl (pH 7.5) containing 50 mM DTT at 37 °C for 20 min.

    Nuclear Magnetic Resonance:

    Article Title: Structure of the Parkin in-between-ring domain provides insights for E3-ligase dysfunction in autosomal recessive Parkinson's disease
    Article Snippet: .. Proteins were dialyzed against 25 mM Na2 HPO4 pH 6.95, 100 mM NaCl, and 1 mM DTT and concentrated to 0.2–0.9 mM by ultrafiltration (Millipore, Mississauga, ON, Canada) for NMR experiments. .. NMR experiments were acquired on a Varian Inova 600 MHz spectrometer using a xyz -gradient triple-resonance probe.

    other:

    Article Title: Synergism between a foldase and an unfoldase: reciprocal dependence between the thioredoxin-like activity of DnaJ and the polypeptide-unfolding activity of DnaK
    Article Snippet: Insulin turbidity assay The insulin turbidity assay was performed as described in Holmgren ( ) with following modifications; Native insulin (0.13 mM) supplemented either with thioredoxin (1 μM and 10 μM), DnaJ (30 μM) or ΔDnaJ (30 μM) in presence of 0.45 mM DTT (Sigma-Aldrich).

    Activity Assay:

    Article Title: Structure of the Neurospora SET Domain Protein DIM-5, a Histone H3 Lysine Methyltransferase
    Article Snippet: .. The activity was assayed in a 20 µl reaction containing 50 mM glycine (pH 9.8), 2 mM DTT, 40–80 µM unlabeled AdoMet (Sigma), 0.5 µCi [methyl-3 H]AdoMet (78 Ci/mmol, NEN NET155H), 0.25–0.5 µg of DIM-5 protein, and 2–5 µg histones (calf thymus histones Sigma H4524, Roche 223565, or recombinant chicken erythrocyte histones, a gift from Dr. V. Ramakrishnan). .. The reaction was incubated at room temperature for 10–15 min and methylation was analyzed either by SDS-PAGE and fluorography or by precipitation with 20% TCA, filtration (Milipore GF/F filter), washing, and liquid scintillation counting.

    Protease Inhibitor:

    Article Title: ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway
    Article Snippet: .. Drug Treatments Working concentrations are 0.1 μM thapsigargin, 0.5 mM dithiothreitol, 5 mM methyl-β-cyclodextrin, and 1 μM brefeldin A. Lysosomal protease inhibitors refer to 125 μM leupeptin + protease inhibitor cocktail (Sigma P8340) diluted 1:100. .. Microscopy All fixed and live-cell imaging, excluding FRAP, were performed using the Marianas spinning disk confocal system (Intelligent Imaging Innovations) attached to a Zeiss Observer.Z1 microscope (Carl Zeiss MicroImaging) and analyzed by Slidebook software.

    Recombinant:

    Article Title: Structure of the Neurospora SET Domain Protein DIM-5, a Histone H3 Lysine Methyltransferase
    Article Snippet: .. The activity was assayed in a 20 µl reaction containing 50 mM glycine (pH 9.8), 2 mM DTT, 40–80 µM unlabeled AdoMet (Sigma), 0.5 µCi [methyl-3 H]AdoMet (78 Ci/mmol, NEN NET155H), 0.25–0.5 µg of DIM-5 protein, and 2–5 µg histones (calf thymus histones Sigma H4524, Roche 223565, or recombinant chicken erythrocyte histones, a gift from Dr. V. Ramakrishnan). .. The reaction was incubated at room temperature for 10–15 min and methylation was analyzed either by SDS-PAGE and fluorography or by precipitation with 20% TCA, filtration (Milipore GF/F filter), washing, and liquid scintillation counting.

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    Millipore dtt
    Shown are 600 MHz 1 H- 15 N HSQC spectra of Parkin IBR 307–384 . ( a ) Folded 0.2 mM IBR 307–384 in 25 mM phosphate buffer (pH 6.95), 100 mM <t>NaCl,</t> 1 mM <t>DTT</t> at 25°C. ( b ) Spectrum of IBR 307–384 from a upon EDTA addition showing the
    Dtt, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sputolysin reagent
    Positive correlation of BPI and IL-8 concentrations in cell-free lung fluid from CF patients. Supernatants of sputum samples from CF patients were collected after treatment with <t>sputolysin</t> and subsequent centrifugation. IL-8 and BPI levels were determined by ELISA. Linear regression shows the positive correlation r 2 = 0.6785. The P value was
    Sputolysin Reagent, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shown are 600 MHz 1 H- 15 N HSQC spectra of Parkin IBR 307–384 . ( a ) Folded 0.2 mM IBR 307–384 in 25 mM phosphate buffer (pH 6.95), 100 mM NaCl, 1 mM DTT at 25°C. ( b ) Spectrum of IBR 307–384 from a upon EDTA addition showing the

    Journal:

    Article Title: Structure of the Parkin in-between-ring domain provides insights for E3-ligase dysfunction in autosomal recessive Parkinson's disease

    doi: 10.1073/pnas.0610548104

    Figure Lengend Snippet: Shown are 600 MHz 1 H- 15 N HSQC spectra of Parkin IBR 307–384 . ( a ) Folded 0.2 mM IBR 307–384 in 25 mM phosphate buffer (pH 6.95), 100 mM NaCl, 1 mM DTT at 25°C. ( b ) Spectrum of IBR 307–384 from a upon EDTA addition showing the

    Article Snippet: Proteins were dialyzed against 25 mM Na2 HPO4 pH 6.95, 100 mM NaCl, and 1 mM DTT and concentrated to 0.2–0.9 mM by ultrafiltration (Millipore, Mississauga, ON, Canada) for NMR experiments.

    Techniques:

    Thiol-redox state of His-Sll1961 protein detected by modification with PEG-maleimide. ( A , B ) WT and cysteine mutants of His-Sll1961 protein were reduced by DTT or oxidized by diamide, modified with PEG-maleimide, fractionated by non-reducing 12% SDS-PAGE and stained with CBB. ( C , D ) Effects of incubation with TrxM on the redox state of Sll1961. WT and cysteine mutants of His-Sll1961 protein oxidized by 500 μM diamide was incubated with DTT (0.1 or 100 mM) or TrxM (0.5 or 5 μM) in the presence of 0.1 mM DTT, modified with PEG-maleimide, fractionated by non-reducing 12% SDS-PAGE and stained with CBB. 0PEG, 1PEG, 2PEG and 3PEG indicate the number of cysteine residues modified with PEG-maleimide.

    Journal: Scientific Reports

    Article Title: Interaction of the GntR-family transcription factor Sll1961 with thioredoxin in the cyanobacterium Synechocystis sp. PCC 6803

    doi: 10.1038/s41598-018-25077-5

    Figure Lengend Snippet: Thiol-redox state of His-Sll1961 protein detected by modification with PEG-maleimide. ( A , B ) WT and cysteine mutants of His-Sll1961 protein were reduced by DTT or oxidized by diamide, modified with PEG-maleimide, fractionated by non-reducing 12% SDS-PAGE and stained with CBB. ( C , D ) Effects of incubation with TrxM on the redox state of Sll1961. WT and cysteine mutants of His-Sll1961 protein oxidized by 500 μM diamide was incubated with DTT (0.1 or 100 mM) or TrxM (0.5 or 5 μM) in the presence of 0.1 mM DTT, modified with PEG-maleimide, fractionated by non-reducing 12% SDS-PAGE and stained with CBB. 0PEG, 1PEG, 2PEG and 3PEG indicate the number of cysteine residues modified with PEG-maleimide.

    Article Snippet: WT and cysteine mutants of His-Sll1961 protein were reduced with 100 mM DTT for 15 min at room temperature, concentrated to 60 μM using Amicon Ultra-0.5 mL (MWCO 10 kDa, Millipore), centrifuged at 20,600 g for 30 min at 4 °C, and filtrated using a 0.45 μm syringe filter (Millipore) before injection.

    Techniques: Modification, SDS Page, Staining, Incubation

    Size exclusion chromatography elution profiles of His-Sll1961 protein. WT and cysteine mutants of His-Sll1961 recombinant proteins were reduced with 100 mM DTT for 15 min at room temperature and applied to a gel filtration column. Vertical dotted lines show the retention times of standard proteins with 150, 66, and 44 kDa.

    Journal: Scientific Reports

    Article Title: Interaction of the GntR-family transcription factor Sll1961 with thioredoxin in the cyanobacterium Synechocystis sp. PCC 6803

    doi: 10.1038/s41598-018-25077-5

    Figure Lengend Snippet: Size exclusion chromatography elution profiles of His-Sll1961 protein. WT and cysteine mutants of His-Sll1961 recombinant proteins were reduced with 100 mM DTT for 15 min at room temperature and applied to a gel filtration column. Vertical dotted lines show the retention times of standard proteins with 150, 66, and 44 kDa.

    Article Snippet: WT and cysteine mutants of His-Sll1961 protein were reduced with 100 mM DTT for 15 min at room temperature, concentrated to 60 μM using Amicon Ultra-0.5 mL (MWCO 10 kDa, Millipore), centrifuged at 20,600 g for 30 min at 4 °C, and filtrated using a 0.45 μm syringe filter (Millipore) before injection.

    Techniques: Size-exclusion Chromatography, Recombinant, Filtration

    Interaction between Sll1961 and TrxM detected in E . coli co-expression strain. ( A ) Detection of the interaction between His-tagged Sll1961 and S-tagged TrxM C35S . After the soluble protein fraction of the control Origami2 strain (Control), the strain expressing only TrxM C35S (Trx), the strain expressing only Sll1961 (Sll1961) and the strain expressing both TrxM C35S and Sll1961 (Trx-Sll1961) was separated by non-reducing 12% SDS-PAGE, Sll1961 and Trx were detected by immunoblot analysis using a His-tag antibody (left) and S-protein (right), respectively. ± indicates with or without 100 mM DTT treatment. Black, gray and white arrow heads indicate the Trx-Sll1961 complex, Sll1961 monomer and Trx monomer, respectively. ( B ) Expression levels of Sll1961 and Trx in the strain expressing only TrxM C35S (Trx) and strains expressing both TrxM C35S and Sll1961 (WT, C124A, C229A, C307A). The soluble fraction of each strain was separated by 12% SDS-PAGE and stained with CBB. ( C ) Effect of cysteine substitutions in Sll1961 on the interaction with TrxM C35S . The soluble fraction of the strains expressing only Sll1961 (WT, C124A, C229A, C307A) and the strains expressing both TrxM C35S and Sll1961 (WT, C124A, C229A, C307A) was separated by non-reducing 12% SDS-PAGE and Sll1961 was detected by immunoblot analysis using a His-tag antibody. ( D ) The soluble fraction of the strain expressing only TrxM C35S (Trx) and the strains expressing both TrxM C35S and Sll1961 (WT, C124A, C229A, C307A) was separated by non-reducing 12% SDS-PAGE and Trx was detected by immunoblot analysis using S-protein. ( E ) The enlarged view of the Trx-Sll1961 complex and the adjacent non-specific band observed in Fig. 1D.

    Journal: Scientific Reports

    Article Title: Interaction of the GntR-family transcription factor Sll1961 with thioredoxin in the cyanobacterium Synechocystis sp. PCC 6803

    doi: 10.1038/s41598-018-25077-5

    Figure Lengend Snippet: Interaction between Sll1961 and TrxM detected in E . coli co-expression strain. ( A ) Detection of the interaction between His-tagged Sll1961 and S-tagged TrxM C35S . After the soluble protein fraction of the control Origami2 strain (Control), the strain expressing only TrxM C35S (Trx), the strain expressing only Sll1961 (Sll1961) and the strain expressing both TrxM C35S and Sll1961 (Trx-Sll1961) was separated by non-reducing 12% SDS-PAGE, Sll1961 and Trx were detected by immunoblot analysis using a His-tag antibody (left) and S-protein (right), respectively. ± indicates with or without 100 mM DTT treatment. Black, gray and white arrow heads indicate the Trx-Sll1961 complex, Sll1961 monomer and Trx monomer, respectively. ( B ) Expression levels of Sll1961 and Trx in the strain expressing only TrxM C35S (Trx) and strains expressing both TrxM C35S and Sll1961 (WT, C124A, C229A, C307A). The soluble fraction of each strain was separated by 12% SDS-PAGE and stained with CBB. ( C ) Effect of cysteine substitutions in Sll1961 on the interaction with TrxM C35S . The soluble fraction of the strains expressing only Sll1961 (WT, C124A, C229A, C307A) and the strains expressing both TrxM C35S and Sll1961 (WT, C124A, C229A, C307A) was separated by non-reducing 12% SDS-PAGE and Sll1961 was detected by immunoblot analysis using a His-tag antibody. ( D ) The soluble fraction of the strain expressing only TrxM C35S (Trx) and the strains expressing both TrxM C35S and Sll1961 (WT, C124A, C229A, C307A) was separated by non-reducing 12% SDS-PAGE and Trx was detected by immunoblot analysis using S-protein. ( E ) The enlarged view of the Trx-Sll1961 complex and the adjacent non-specific band observed in Fig. 1D.

    Article Snippet: WT and cysteine mutants of His-Sll1961 protein were reduced with 100 mM DTT for 15 min at room temperature, concentrated to 60 μM using Amicon Ultra-0.5 mL (MWCO 10 kDa, Millipore), centrifuged at 20,600 g for 30 min at 4 °C, and filtrated using a 0.45 μm syringe filter (Millipore) before injection.

    Techniques: Expressing, SDS Page, Staining

    Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with DLB (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by UCH-L1 in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.

    Journal: The American Journal of Pathology

    Article Title: Ubiquitination of ?-Synuclein Is Not Required for Formation of Pathological Inclusions in ?-Synucleinopathies

    doi:

    Figure Lengend Snippet: Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with DLB (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction ( lane 1 ), TX fraction ( lane 2 ), sarkosyl-soluble fraction ( lane 3 ), and SDS-soluble fraction ( lane 4 ) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 ( arrows , mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by UCH-L1 in vitro . SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.

    Article Snippet: SDS-soluble fractions from the cingulate cortex of neuropathologically normal (NL) or DLB brains were diluted 40-fold in UCH buffer (50 mmol/L HEPES, pH 7.8, 0.5 mmol/L EDTA, 1 mmol/L dithiothreitol) and then concentrated 40-fold using a MicroconYM-10 (Millipore Corp., Bedford, MA) to remove SDS.

    Techniques: Western Blot, Recombinant, Immunoprecipitation, Isolation, Modification, In Vitro

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

    Journal: Infection and Immunity

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

    doi: 10.1128/IAI.02066-05

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

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

    Techniques: Centrifugation, Enzyme-linked Immunosorbent Assay