dtt  (Thermo Fisher)


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  • 90
    Name:
    Dithiothreitol DTT
    Description:
    Disufide crosslinks of cystines in proteins can be reduced to cysteine residues by dithiothreitol DTT
    Catalog Number:
    d1532
    Price:
    None
    Applications:
    Cell Analysis|Labeling Thiols|Other Thiol-Reactive Probes|Protein, Peptide & Antibody Labeling|Labeling Chemistry
    Category:
    Lab Reagents and Chemicals
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    Structured Review

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

    1) Product Images from "The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism"

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

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E13-11-0658

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

    Techniques Used: Mutagenesis, Transfection, Avidin-Biotin Assay

    2) Product Images from "Induced Expression of Drug Metabolizing Enzymes by Preventive Agents: Role of the Antioxidant Response Element"

    Article Title: Induced Expression of Drug Metabolizing Enzymes by Preventive Agents: Role of the Antioxidant Response Element

    Journal: Chemico-biological interactions

    doi: 10.1016/j.cbi.2009.08.011

    Gene Cluster Analysis of Both Phase I and Phase II Enzymes. Group A: genes preferentially induced by PB, DAS and EXO; Group B: genes induced by DTT; Group C: genes induced by BF; Group D: genes induced by DAS and DTT.
    Figure Legend Snippet: Gene Cluster Analysis of Both Phase I and Phase II Enzymes. Group A: genes preferentially induced by PB, DAS and EXO; Group B: genes induced by DTT; Group C: genes induced by BF; Group D: genes induced by DAS and DTT.

    Techniques Used:

    3) 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

    Primary tissue culture cells from newly-resected gliomas also display inducible elements of the UPR. Dissociated cell cultured from freshly-resected GBMs were grown under serum-free conditions and were treated (or not, “Cont”) with 1 mM DTT (“+DTT”) for 4 hrs. Cell cultures were harvested, and cells lysed described. Proteins were separated on SDS-PAGE and Western blotted and probed with the antibodies listed. Upregulation of some of the UPR components is evident. Actin probe is used as a loading control, from the stripped CRT blot. Other actin blots to verify loading are shown in Supplemental Figure S3 .
    Figure Legend Snippet: Primary tissue culture cells from newly-resected gliomas also display inducible elements of the UPR. Dissociated cell cultured from freshly-resected GBMs were grown under serum-free conditions and were treated (or not, “Cont”) with 1 mM DTT (“+DTT”) for 4 hrs. Cell cultures were harvested, and cells lysed described. Proteins were separated on SDS-PAGE and Western blotted and probed with the antibodies listed. Upregulation of some of the UPR components is evident. Actin probe is used as a loading control, from the stripped CRT blot. Other actin blots to verify loading are shown in Supplemental Figure S3 .

    Techniques Used: Cell Culture, SDS Page, Western Blot

    U87MG cells following UPR induction are resistant to temozolomide in clonogenic assays. U87 cells were either left untreated or stressed with 1 mM DTT (UPR inducer) for 4 hrs. Cells were then treated (or with vehicle control) with 0.2 or 1.0 µM temozolomide (TMZ) for 24 hrs. Cells were then washed and plated in soft agar for clonogenicity assays until a minimum of colonies of > 50 cells could be identified, which were then counted. Average colony counts with standard deviations are shown; ANOVA results of significant differences between groups (p
    Figure Legend Snippet: U87MG cells following UPR induction are resistant to temozolomide in clonogenic assays. U87 cells were either left untreated or stressed with 1 mM DTT (UPR inducer) for 4 hrs. Cells were then treated (or with vehicle control) with 0.2 or 1.0 µM temozolomide (TMZ) for 24 hrs. Cells were then washed and plated in soft agar for clonogenicity assays until a minimum of colonies of > 50 cells could be identified, which were then counted. Average colony counts with standard deviations are shown; ANOVA results of significant differences between groups (p

    Techniques Used:

    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

    Identification of UPR signaling response patterns in high-grade glioma xenografts and cell lines. Human glioma xenografts grown in nu/nu mice were derived from U87MG, and U87+EGFR (wild type) (cell lines described in the text and Materials and Methods). ( A ) Northern blots of 10 µg total RNA from replicate tumors (n=3) and normal brain from nu/nu mice; 10 µg total RNA from U87 tissue culture cells (“cells”) treated with the reducing agent DTT ([+]) lanes) to induce the UPR. Note transcriptional upregulation of UPR-induced mRNAs for ER chaperones (GRP94, BiP/GRP78) and UPR signaling components (XBP-1, CHOP, ATF4, ATF6). Quantification of BiP/GRP78 ( B ) and GRP94 ( C ) mRNA expression compared to mean level of expression in normal murine brain (dotted line). ( D ) U87MG, U87+EGFR, and U87+EGFRvIII (U87 cells transfected with the tumor-specific EGFR mutant variant III [in-frame deletion of exons 2-7]) cells show greater UPR inducibility with 1 mM DTT (determined by Northern blotting for XBP-1 and CHOP messages) than do HeLa cells. ( E ) Human glioma xenografts were derived from U87MG, U87+EGFR, and from D245MG, from a patient-derived Duke high grade glioma (from the Duke Brain Tumor BioRepository). Immunoblot of replicate tumors (n=3) from xenograft glioma models and normal brain from nu/nu mice. Note upregulation of ER chaperones in tumor lysates vs brain lysates: GRP170/ORP150, GRP94, calnexin (CNX), ERp72, protein disulfide isomerase (PDI), calreticulin (CRT), homocysteine-induced ER protein (HERP), and ER membrane markers (Sec61α and translocon associated protein, TRAPα) relative to loading control (β-actin). GRP78/BiP protein expression was variable in our Western blot assays. Blots probing for actin as loading controls are found in Supplemental Figure S1 . Blots for GRPs 170 and 78, for ERp72 and TRAPα were replicate blots. Blots for GRP94, CNX, CRT, HERP, and Sec61α were stripped and reprobed for actin.
    Figure Legend Snippet: Identification of UPR signaling response patterns in high-grade glioma xenografts and cell lines. Human glioma xenografts grown in nu/nu mice were derived from U87MG, and U87+EGFR (wild type) (cell lines described in the text and Materials and Methods). ( A ) Northern blots of 10 µg total RNA from replicate tumors (n=3) and normal brain from nu/nu mice; 10 µg total RNA from U87 tissue culture cells (“cells”) treated with the reducing agent DTT ([+]) lanes) to induce the UPR. Note transcriptional upregulation of UPR-induced mRNAs for ER chaperones (GRP94, BiP/GRP78) and UPR signaling components (XBP-1, CHOP, ATF4, ATF6). Quantification of BiP/GRP78 ( B ) and GRP94 ( C ) mRNA expression compared to mean level of expression in normal murine brain (dotted line). ( D ) U87MG, U87+EGFR, and U87+EGFRvIII (U87 cells transfected with the tumor-specific EGFR mutant variant III [in-frame deletion of exons 2-7]) cells show greater UPR inducibility with 1 mM DTT (determined by Northern blotting for XBP-1 and CHOP messages) than do HeLa cells. ( E ) Human glioma xenografts were derived from U87MG, U87+EGFR, and from D245MG, from a patient-derived Duke high grade glioma (from the Duke Brain Tumor BioRepository). Immunoblot of replicate tumors (n=3) from xenograft glioma models and normal brain from nu/nu mice. Note upregulation of ER chaperones in tumor lysates vs brain lysates: GRP170/ORP150, GRP94, calnexin (CNX), ERp72, protein disulfide isomerase (PDI), calreticulin (CRT), homocysteine-induced ER protein (HERP), and ER membrane markers (Sec61α and translocon associated protein, TRAPα) relative to loading control (β-actin). GRP78/BiP protein expression was variable in our Western blot assays. Blots probing for actin as loading controls are found in Supplemental Figure S1 . Blots for GRPs 170 and 78, for ERp72 and TRAPα were replicate blots. Blots for GRP94, CNX, CRT, HERP, and Sec61α were stripped and reprobed for actin.

    Techniques Used: Mouse Assay, Derivative Assay, Northern Blot, Expressing, Transfection, Mutagenesis, Variant Assay, Western Blot

    cDNA microarray analysis of polyribosome-engaged transcripts in control and UPR-activated cells and xenograft glioma tumors. ( A ) Polyribosome traces from U87MG glioma models experiencing (or not) various stresses: unstressed cells, DTT-treated (acutely) stressed cells, and xenograft tumor-derived samples (“ in vivo chronic stressed tumor”). Each sample was analyzed in triplicate. Following homogenization, sample lysates were layered over a linear sucrose gradient (15-50%), separated at 150,000X g for 3 hours and processed as described ( Figure S2 ). Polyribosome-containing regions were pooled, total RNA extracted and submitted to the Duke Microarray Facility for analysis. ( B ) Heatmap of triplicate samples with one-way ANOVA analysis (P
    Figure Legend Snippet: cDNA microarray analysis of polyribosome-engaged transcripts in control and UPR-activated cells and xenograft glioma tumors. ( A ) Polyribosome traces from U87MG glioma models experiencing (or not) various stresses: unstressed cells, DTT-treated (acutely) stressed cells, and xenograft tumor-derived samples (“ in vivo chronic stressed tumor”). Each sample was analyzed in triplicate. Following homogenization, sample lysates were layered over a linear sucrose gradient (15-50%), separated at 150,000X g for 3 hours and processed as described ( Figure S2 ). Polyribosome-containing regions were pooled, total RNA extracted and submitted to the Duke Microarray Facility for analysis. ( B ) Heatmap of triplicate samples with one-way ANOVA analysis (P

    Techniques Used: Microarray, Derivative Assay, In Vivo, Homogenization

    Relative metabolomic outputs of U87MG cells subjected to UPR stress compared to unstressed cells. U87 cells were grown in Knockout DMEM medium with serum replacement as described above. Cells were harvested, washed, and replated in the same (fresh) medium with or without 1 mM DTT, and with 5 mM 13 C-glucose, for 4 hrs prior to cell and media harvest and PCA extraction as described in Materials and Methods. 1 H-, 31 P-, and 13 C-NMR spectra were obtained and quantified; data analyses were conducted as described. Graphs compare metabolite components from untreated cells (set to 100%) vs treated cells; error bars show standard deviation, and * = p
    Figure Legend Snippet: Relative metabolomic outputs of U87MG cells subjected to UPR stress compared to unstressed cells. U87 cells were grown in Knockout DMEM medium with serum replacement as described above. Cells were harvested, washed, and replated in the same (fresh) medium with or without 1 mM DTT, and with 5 mM 13 C-glucose, for 4 hrs prior to cell and media harvest and PCA extraction as described in Materials and Methods. 1 H-, 31 P-, and 13 C-NMR spectra were obtained and quantified; data analyses were conducted as described. Graphs compare metabolite components from untreated cells (set to 100%) vs treated cells; error bars show standard deviation, and * = p

    Techniques Used: Knock-Out, Nuclear Magnetic Resonance, Standard Deviation

    4) Product Images from "H2O2 dynamics in the malaria parasite Plasmodium falciparum"

    Article Title: H2O2 dynamics in the malaria parasite Plasmodium falciparum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174837

    Dynamic range of roGFP2-Orp1 and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM DTT at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p
    Figure Legend Snippet: Dynamic range of roGFP2-Orp1 and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM DTT at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p

    Techniques Used: Transfection, Lysis, Confocal Laser Scanning Microscopy, Fluorescence, Incubation

    5) Product Images from "Neuronal Microtubule-associated Protein 2D Is a Dual A-kinase Anchoring Protein Expressed in Rat Ovarian Granulosa Cells *"

    Article Title: Neuronal Microtubule-associated Protein 2D Is a Dual A-kinase Anchoring Protein Expressed in Rat Ovarian Granulosa Cells *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M402980200

    DEAE-cellulose and cAMP-agarose affinity chromatography of PO ovarian extracts A , detergent-soluble ovarian extracts (800 μg of protein in 300 μl) were prepared (without DTT), subjected to cross-linking by incubating with 1 mM DSP for 15 min at room temperature, and then subjected to immunoprecipitation using the indicated antibodies (anti-MAP2 (Sigma), anti-RI (BD Biosciences), and anti-RII (Upstate Biotechnology)). Flow-through ( FT ) represents 27% of the extract that was not pulled down by the antibody-agarose complex. IP , immunoprecipitated complex. Samples were subjected to SDS-PAGE and blotted with anti-MAP2 antibody. Results for RII and NI immunoprecipitations are overexposed to confirm the absence of signal in the immunoprecipitation complex lanes. Results are representative of three separate experiments. B , ovaries from 15 rats were harvested 48 h post-PMSG injection and homogenized in buffer E, and a soluble extract was prepared by centrifuging at 105,000 × g for 15 min and loading onto a DEAE-cellulose column. Proteins were eluted with a linear salt gradient, collecting fractions in buffer B but without DTT. Aliquots of odd-numbered fractions were mixed with SDS-PAGE sample buffer, boiled, and subjected to SDS-PAGE and Western blotting with the indicated antibodies. Results are representative of four separate experiments. C , a graphic representation of the Western data presented in B but now normalized to percentage of maximal signal. Also shown is the cAMP-stimulated PKA activity, also normalized to percentage of maximal signal ( lower portion of C ). In D , fractions from the indicated DEAE-cellulose peak fractions (shown in C ) were pooled, concentrated, and incubated with cAMP agarose. The agarose was washed with low and high salt buffers to remove nonspecifically bound proteins, and the final wash ( FW ) was collected. Specifically bound AKAPs were eluted first with 5 μM Ht31 ( Ht31 ) and then with 75 mM cAMP. The samples were mixed with SDS-PAGE sample buffer and boiled, and aliquots were then subjected to SDS-PAGE and Western blotting using the antibodies indicated. Results are representative of four separate experiments. E , fractions from indicated regions of the DEAE-cellulose column (shown in C ) were pooled, 1-ml aliquots were incubated with the protein cross-linker (DSP; 1 μM) for 15 min at room temperature, and samples were subjected to immunoprecipitation with the indicated antibodies (see A ). A 100-μl aliquot of the 1-ml starting material was boiled and applied to the gel ( Load ). Agarose pellets were washed with buffer D, and proteins were eluted from the protein A+G-agarose with 150 μl of Immunopure® elution buffer, pH 2.8, mixed with SDS-PAGE sample buffer, and boiled. 67% of the total eluate ( IP ) was loaded onto the gel for SDS-PAGE. Results are representative of two separate experiments. F , immunoprecipitations from pooled and concentrated DEAE peak 1 and 2 fractions ( lanes 1 ) or from PKA IIβ holoenzyme in these fractions that was sedimented by sucrose density gradient centrifugation ( lanes 3 ) were conducted with Sigma anti-MAP2 antibody, anti-HA (NI), or BD Biosciences anti-RIIβ antibodies. Lanes 1 and 2 , immunodepletion results for proteins that were not imunoprecipitated. Lanes 3 and 4 , immunoprecipitation results. Results are representative of two independent experiments.
    Figure Legend Snippet: DEAE-cellulose and cAMP-agarose affinity chromatography of PO ovarian extracts A , detergent-soluble ovarian extracts (800 μg of protein in 300 μl) were prepared (without DTT), subjected to cross-linking by incubating with 1 mM DSP for 15 min at room temperature, and then subjected to immunoprecipitation using the indicated antibodies (anti-MAP2 (Sigma), anti-RI (BD Biosciences), and anti-RII (Upstate Biotechnology)). Flow-through ( FT ) represents 27% of the extract that was not pulled down by the antibody-agarose complex. IP , immunoprecipitated complex. Samples were subjected to SDS-PAGE and blotted with anti-MAP2 antibody. Results for RII and NI immunoprecipitations are overexposed to confirm the absence of signal in the immunoprecipitation complex lanes. Results are representative of three separate experiments. B , ovaries from 15 rats were harvested 48 h post-PMSG injection and homogenized in buffer E, and a soluble extract was prepared by centrifuging at 105,000 × g for 15 min and loading onto a DEAE-cellulose column. Proteins were eluted with a linear salt gradient, collecting fractions in buffer B but without DTT. Aliquots of odd-numbered fractions were mixed with SDS-PAGE sample buffer, boiled, and subjected to SDS-PAGE and Western blotting with the indicated antibodies. Results are representative of four separate experiments. C , a graphic representation of the Western data presented in B but now normalized to percentage of maximal signal. Also shown is the cAMP-stimulated PKA activity, also normalized to percentage of maximal signal ( lower portion of C ). In D , fractions from the indicated DEAE-cellulose peak fractions (shown in C ) were pooled, concentrated, and incubated with cAMP agarose. The agarose was washed with low and high salt buffers to remove nonspecifically bound proteins, and the final wash ( FW ) was collected. Specifically bound AKAPs were eluted first with 5 μM Ht31 ( Ht31 ) and then with 75 mM cAMP. The samples were mixed with SDS-PAGE sample buffer and boiled, and aliquots were then subjected to SDS-PAGE and Western blotting using the antibodies indicated. Results are representative of four separate experiments. E , fractions from indicated regions of the DEAE-cellulose column (shown in C ) were pooled, 1-ml aliquots were incubated with the protein cross-linker (DSP; 1 μM) for 15 min at room temperature, and samples were subjected to immunoprecipitation with the indicated antibodies (see A ). A 100-μl aliquot of the 1-ml starting material was boiled and applied to the gel ( Load ). Agarose pellets were washed with buffer D, and proteins were eluted from the protein A+G-agarose with 150 μl of Immunopure® elution buffer, pH 2.8, mixed with SDS-PAGE sample buffer, and boiled. 67% of the total eluate ( IP ) was loaded onto the gel for SDS-PAGE. Results are representative of two separate experiments. F , immunoprecipitations from pooled and concentrated DEAE peak 1 and 2 fractions ( lanes 1 ) or from PKA IIβ holoenzyme in these fractions that was sedimented by sucrose density gradient centrifugation ( lanes 3 ) were conducted with Sigma anti-MAP2 antibody, anti-HA (NI), or BD Biosciences anti-RIIβ antibodies. Lanes 1 and 2 , immunodepletion results for proteins that were not imunoprecipitated. Lanes 3 and 4 , immunoprecipitation results. Results are representative of two independent experiments.

    Techniques Used: Affinity Chromatography, Immunoprecipitation, Flow Cytometry, SDS Page, Injection, Western Blot, Activity Assay, Incubation, Gradient Centrifugation

    6) Product Images from "Hydrogen peroxide dynamics in subcellular compartments of malaria parasites using genetically encoded redox probes"

    Article Title: Hydrogen peroxide dynamics in subcellular compartments of malaria parasites using genetically encoded redox probes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10093-8

    Dynamic range of roGFP2-Orp1 and Mito-roGFP2-Orp1 in transfected NF54- attB parasites. NF54- attB parasites transfected with roGFP2-Orp1 or Mito-roGFP2-Orp1 were exposed to 1 mM DIA or 10 mM DTT for 2 min before blocking with 2 mM NEM. Fluorescence ratios of 405/488 nm were detected with CLSM. NF54 [roGFP2-Orp1] - attB parasites showed a slightly higher DIA sensitivity than NF54 [Mito-roGFP2-Orp1] - attB parasites. CLSM data were composed of values from at least 10–20 trophozoites analyzed per experiment. Mean values and standard errors of the means (±SEM) are shown for three independent experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (***p
    Figure Legend Snippet: Dynamic range of roGFP2-Orp1 and Mito-roGFP2-Orp1 in transfected NF54- attB parasites. NF54- attB parasites transfected with roGFP2-Orp1 or Mito-roGFP2-Orp1 were exposed to 1 mM DIA or 10 mM DTT for 2 min before blocking with 2 mM NEM. Fluorescence ratios of 405/488 nm were detected with CLSM. NF54 [roGFP2-Orp1] - attB parasites showed a slightly higher DIA sensitivity than NF54 [Mito-roGFP2-Orp1] - attB parasites. CLSM data were composed of values from at least 10–20 trophozoites analyzed per experiment. Mean values and standard errors of the means (±SEM) are shown for three independent experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (***p

    Techniques Used: Transfection, Blocking Assay, Fluorescence, Confocal Laser Scanning Microscopy

    7) Product Images from "Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro"

    Article Title: Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro

    Journal: Biology Open

    doi: 10.1242/bio.20149340

    CD spectra of purified human Rab GTPases. Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl 2 (5 mM), and DTT (1 mM).
    Figure Legend Snippet: CD spectra of purified human Rab GTPases. Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl 2 (5 mM), and DTT (1 mM).

    Techniques Used: Purification

    8) Product Images from "Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway"

    Article Title: Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

    Journal: Redox Biology

    doi: 10.1016/j.redox.2019.101400

    BQ-induced enzyme inactivation and covalent modification of purified Trx1 protein. (A) Concentration-dependent inhibition of Trx1 activity by BQ. DTT-reduced Trx1 (2.5 μM) was incubated with BQ (0.5–10 μM) in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), with Trx1 activity determined after 5 min of treatment. (B) BQ-induced quinoprotein detection. DTT-reduced Trx1 (10 μM) was incubated with BQ (5–40 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed by SDS-PAGE followed by NBT redox staining. (C) Free thiol detection using BEI labelling. DTT-reduced Trx1 (5 μM) was incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by incubation with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection. * p
    Figure Legend Snippet: BQ-induced enzyme inactivation and covalent modification of purified Trx1 protein. (A) Concentration-dependent inhibition of Trx1 activity by BQ. DTT-reduced Trx1 (2.5 μM) was incubated with BQ (0.5–10 μM) in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), with Trx1 activity determined after 5 min of treatment. (B) BQ-induced quinoprotein detection. DTT-reduced Trx1 (10 μM) was incubated with BQ (5–40 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed by SDS-PAGE followed by NBT redox staining. (C) Free thiol detection using BEI labelling. DTT-reduced Trx1 (5 μM) was incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by incubation with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection. * p

    Techniques Used: Modification, Purification, Concentration Assay, Inhibition, Activity Assay, Incubation, SDS Page, Staining

    Identification of BQ-modified Cys residues in Trx1. DTT-reduced Trx1 (10 μM) was incubated with BQ (50 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The BQ-treated Trx1 was then digested using Lys-C at 37 °C overnight and analysed by LC-MS/MS (seen Materials and methods for further details). (A) Peptide 1, (B) Peptide 2, (C) Peptide 3 (cf. Table 1 ).
    Figure Legend Snippet: Identification of BQ-modified Cys residues in Trx1. DTT-reduced Trx1 (10 μM) was incubated with BQ (50 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The BQ-treated Trx1 was then digested using Lys-C at 37 °C overnight and analysed by LC-MS/MS (seen Materials and methods for further details). (A) Peptide 1, (B) Peptide 2, (C) Peptide 3 (cf. Table 1 ).

    Techniques Used: Modification, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Effect of GSH on BQ-induced enzyme activity loss, protein cross-linking and modification. Panels A, C, E: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) in the presence of GSH (5–1000 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The samples were split into three portions. The remaining Trx activity was measured (panel A), samples were analysed by SDS-PAGE followed by silver staining (panel C), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel E). Panels B, D, F: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), then GSH (5–1000 μM) was added and incubated for another 5 min. The samples were then split into three portions and analysed, as described above, for remaining Trx activity (panel B), by SDS-PAGE followed by silver staining (panel D), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel F). Data (mean ± standard deviations) from 3 independent experiments are presented in panels A and B. Representative images from 3 independent experiments are shown in panels (C - F). * p
    Figure Legend Snippet: Effect of GSH on BQ-induced enzyme activity loss, protein cross-linking and modification. Panels A, C, E: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) in the presence of GSH (5–1000 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The samples were split into three portions. The remaining Trx activity was measured (panel A), samples were analysed by SDS-PAGE followed by silver staining (panel C), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel E). Panels B, D, F: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), then GSH (5–1000 μM) was added and incubated for another 5 min. The samples were then split into three portions and analysed, as described above, for remaining Trx activity (panel B), by SDS-PAGE followed by silver staining (panel D), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel F). Data (mean ± standard deviations) from 3 independent experiments are presented in panels A and B. Representative images from 3 independent experiments are shown in panels (C - F). * p

    Techniques Used: Activity Assay, Modification, Incubation, SDS Page, Silver Staining

    BQ-induced protein cross-linking. Human Trx1 (5 μM, A and B) and E. coli Trx (5 μM, C and D), pre-treated with DTT, were incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed under non-reducing (A and C) and reducing (B and D) conditions, and then analysed by SDS-PAGE with silver staining. Representative images from 3 independent experiments are shown in each panel.
    Figure Legend Snippet: BQ-induced protein cross-linking. Human Trx1 (5 μM, A and B) and E. coli Trx (5 μM, C and D), pre-treated with DTT, were incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed under non-reducing (A and C) and reducing (B and D) conditions, and then analysed by SDS-PAGE with silver staining. Representative images from 3 independent experiments are shown in each panel.

    Techniques Used: Incubation, SDS Page, Silver Staining

    9) Product Images from "Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation"

    Article Title: Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1703161114

    RLC phosphorylation reduces the number of Myo2 heads bound to actin filaments. Panels show maximum projections of representative fields (128 μm × 128 μm × 50 s) at different KCl concentrations ( A – C ). Phosphorylated Myo2 is shown on the Left (red) and unphosphorylated is shown on the Right ) are shown as averages of multiple fields (N, number of fields). Myo2 was attached to neutravidin-coated coverslips through the C-terminal biotin tag. Conditions were as follows: 30 °C, 25 mM imidazole, pH 7.4, KCl as indicated, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. No methylcellulose was used to allow the diffusion of actin away from the surface. When indicated, Tpm was added to a final concentration of 2 μM. Data are from two independent preparations of Myo2 (N-FLAG-C-Biotin). Error, ± SD.
    Figure Legend Snippet: RLC phosphorylation reduces the number of Myo2 heads bound to actin filaments. Panels show maximum projections of representative fields (128 μm × 128 μm × 50 s) at different KCl concentrations ( A – C ). Phosphorylated Myo2 is shown on the Left (red) and unphosphorylated is shown on the Right ) are shown as averages of multiple fields (N, number of fields). Myo2 was attached to neutravidin-coated coverslips through the C-terminal biotin tag. Conditions were as follows: 30 °C, 25 mM imidazole, pH 7.4, KCl as indicated, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. No methylcellulose was used to allow the diffusion of actin away from the surface. When indicated, Tpm was added to a final concentration of 2 μM. Data are from two independent preparations of Myo2 (N-FLAG-C-Biotin). Error, ± SD.

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Effects of RLC phosphorylation on ATPase activity and motility. ( A ) Steady-state ATPase of unphosphorylated (blue) or phosphorylated (red) Myo2 in the absence or presence of tropomyosin (Tpm). Circles and squares represent independent preparations of N-FLAG-Myo2-C-Biotin. Diamonds represent N-FLAG-Myo2. Conditions were as follows: 30 °C, 10 mM imidazole, pH 7.0, 50 mM NaCl, 1 mM MgCl 2 , 1 mM ATP, and 2 mM DTT. Tpm was added at a 2:1 Actin-Tpm molar ratio. ( B ) In vitro motility speeds of unphosphorylated or phosphorylated Myo2, in the absence or presence of Tpm. Speeds represent motility data from two experimental repeats conducted in parallel with two independent preparations of N-FLAG-Myo2-C-Biotin (n, number of moving filaments). To ensure that all heads were available for interaction with actin, Myo2 was attached to neutravidin-coated coverslips by a biotin tag at its C terminus. Conditions were as follows: 30 °C, 0.5% methylcellulose, 25 mM imidazole, pH 7.4, 50 mM KCl, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. When indicated, Tpm was added to a final concentration of 2 μM.
    Figure Legend Snippet: Effects of RLC phosphorylation on ATPase activity and motility. ( A ) Steady-state ATPase of unphosphorylated (blue) or phosphorylated (red) Myo2 in the absence or presence of tropomyosin (Tpm). Circles and squares represent independent preparations of N-FLAG-Myo2-C-Biotin. Diamonds represent N-FLAG-Myo2. Conditions were as follows: 30 °C, 10 mM imidazole, pH 7.0, 50 mM NaCl, 1 mM MgCl 2 , 1 mM ATP, and 2 mM DTT. Tpm was added at a 2:1 Actin-Tpm molar ratio. ( B ) In vitro motility speeds of unphosphorylated or phosphorylated Myo2, in the absence or presence of Tpm. Speeds represent motility data from two experimental repeats conducted in parallel with two independent preparations of N-FLAG-Myo2-C-Biotin (n, number of moving filaments). To ensure that all heads were available for interaction with actin, Myo2 was attached to neutravidin-coated coverslips by a biotin tag at its C terminus. Conditions were as follows: 30 °C, 0.5% methylcellulose, 25 mM imidazole, pH 7.4, 50 mM KCl, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. When indicated, Tpm was added to a final concentration of 2 μM.

    Techniques Used: Activity Assay, In Vitro, Concentration Assay

    Myo2 does not have the propensity to assemble into filaments. ( A ) Diagram illustrating the domain structure of Myo2 and the high number of Pro residues interspersed throughout the tail. The invariant proline marks the junction between the lever arm and the tail. ( B ) Electron micrographs of platinum shadowed Myo2 (C-Biotin-C-FLAG) molecules showing that Myo2 has two heads and an ∼80-nm-long tail. Filled yellow arrowhead points to a motor domain. Image courtesy of Roger Craig. ( C ) comparing the tails of Myo2 and myosin-II from other species ( A. castellanii , D. discoideum , and human nonmuscle myosin-IIA). Plots begin with the invariant proline and end at the final residue. ( D ) Sedimentation velocity of unphosphorylated or phosphorylated N-FLAG-Myo2 by analytical ultracentrifugation at varying KCl concentrations. A repeat analytical ultracentrifugation experiment with unphosphorylated Myo2 in 150 mM KCl gave a sedimentation coefficient of 7.0S. Conditions were as follows: 20 °C, 10 mM imidazole, pH 7.0, 5 mM MgCl 2 , 1 mM EGTA, 1 mM DTT (KCl as indicated). OD, optical density; S, Svedberg.
    Figure Legend Snippet: Myo2 does not have the propensity to assemble into filaments. ( A ) Diagram illustrating the domain structure of Myo2 and the high number of Pro residues interspersed throughout the tail. The invariant proline marks the junction between the lever arm and the tail. ( B ) Electron micrographs of platinum shadowed Myo2 (C-Biotin-C-FLAG) molecules showing that Myo2 has two heads and an ∼80-nm-long tail. Filled yellow arrowhead points to a motor domain. Image courtesy of Roger Craig. ( C ) comparing the tails of Myo2 and myosin-II from other species ( A. castellanii , D. discoideum , and human nonmuscle myosin-IIA). Plots begin with the invariant proline and end at the final residue. ( D ) Sedimentation velocity of unphosphorylated or phosphorylated N-FLAG-Myo2 by analytical ultracentrifugation at varying KCl concentrations. A repeat analytical ultracentrifugation experiment with unphosphorylated Myo2 in 150 mM KCl gave a sedimentation coefficient of 7.0S. Conditions were as follows: 20 °C, 10 mM imidazole, pH 7.0, 5 mM MgCl 2 , 1 mM EGTA, 1 mM DTT (KCl as indicated). OD, optical density; S, Svedberg.

    Techniques Used: Sedimentation

    Rng3 promotes motility in vitro by binding actin filaments nonspecifically. ( A ) SDS gel showing totals (T), supernatants (S), and pellets (P) from a cosedimentation assay in which 700 nM Rng3 and 75 μM actin were pelleted at 400,000 × g for 20 min in 25 mM imidazole, pH 7.4, 46 mM KCl, 23 mM NaCl, 1 mM EGTA, 4 mM MgCl 2 , and 2 mM DTT. Rng3 does not pellet in the absence of actin. ( B ). Motility assay conditions were as follows: 30 °C, 25 mM imidazole, pH 7.4, 50 mM KCl, 1 mM EGTA, 4 mM MgCl 2 , 10 mM DTT, and 1 mM ATP. Error, ± SD.
    Figure Legend Snippet: Rng3 promotes motility in vitro by binding actin filaments nonspecifically. ( A ) SDS gel showing totals (T), supernatants (S), and pellets (P) from a cosedimentation assay in which 700 nM Rng3 and 75 μM actin were pelleted at 400,000 × g for 20 min in 25 mM imidazole, pH 7.4, 46 mM KCl, 23 mM NaCl, 1 mM EGTA, 4 mM MgCl 2 , and 2 mM DTT. Rng3 does not pellet in the absence of actin. ( B ). Motility assay conditions were as follows: 30 °C, 25 mM imidazole, pH 7.4, 50 mM KCl, 1 mM EGTA, 4 mM MgCl 2 , 10 mM DTT, and 1 mM ATP. Error, ± SD.

    Techniques Used: In Vitro, Binding Assay, SDS-Gel, Motility Assay

    Unphosphorylated Myo2 has motor activity. ( A ) SDS gels of Myo2 heavy chain (HC) coexpressed with its native light chains, Rlc1 (RLC) and Cdc4 (ELC) in the baculovirus/insect cell expression system. Lane 1, molecular mass standards; lane 2, N-FLAG-Myo2; lane 3, Myo2-C-Biotin-C-FLAG; lane 4, N-FLAG-Myo2-C-Biotin. Asterisks (*) indicate breakdown products of the myosin tail. ( B ) Sedimentation velocity of Myo2 by analytical ultracentrifugation. Conditions were as follows: 20 °C, 10 mM imidazole, pH 7.0, 0.5 M KCl, 5 mM MgCl 2 , 1 mM EGTA, 1 mM DTT. OD, optical density; S, Svedberg. ( C ) SDS gel double-stained with phosphoprotein stain and then Coomassie to stain for total protein. The RLC of expressed N-FLAG-Myo2-C-Biotin has the same level of staining as the unphosphorylated smooth muscle myosin standard. ( D ) Rates of steady-state ATPase of Myo2 at various actin concentrations. Circles, squares, and triangles represent independent preparations of N-FLAG-Myo2-C-Biotin. Conditions were as follows: 30 °C, 10 mM imidazole, pH 7.0, 50 mM NaCl, 1 mM MgCl 2 , 1 mM ATP, and 2 mM DTT. ( E ) In vitro motility assays. ( Left ) showing trails of gliding actin filaments. ( Right ) Speeds of actin filament movement (n, number of moving filaments) from 11 experimental repeats with four independent preparations of Myo2 (all three tag variations). Error, ± SD.
    Figure Legend Snippet: Unphosphorylated Myo2 has motor activity. ( A ) SDS gels of Myo2 heavy chain (HC) coexpressed with its native light chains, Rlc1 (RLC) and Cdc4 (ELC) in the baculovirus/insect cell expression system. Lane 1, molecular mass standards; lane 2, N-FLAG-Myo2; lane 3, Myo2-C-Biotin-C-FLAG; lane 4, N-FLAG-Myo2-C-Biotin. Asterisks (*) indicate breakdown products of the myosin tail. ( B ) Sedimentation velocity of Myo2 by analytical ultracentrifugation. Conditions were as follows: 20 °C, 10 mM imidazole, pH 7.0, 0.5 M KCl, 5 mM MgCl 2 , 1 mM EGTA, 1 mM DTT. OD, optical density; S, Svedberg. ( C ) SDS gel double-stained with phosphoprotein stain and then Coomassie to stain for total protein. The RLC of expressed N-FLAG-Myo2-C-Biotin has the same level of staining as the unphosphorylated smooth muscle myosin standard. ( D ) Rates of steady-state ATPase of Myo2 at various actin concentrations. Circles, squares, and triangles represent independent preparations of N-FLAG-Myo2-C-Biotin. Conditions were as follows: 30 °C, 10 mM imidazole, pH 7.0, 50 mM NaCl, 1 mM MgCl 2 , 1 mM ATP, and 2 mM DTT. ( E ) In vitro motility assays. ( Left ) showing trails of gliding actin filaments. ( Right ) Speeds of actin filament movement (n, number of moving filaments) from 11 experimental repeats with four independent preparations of Myo2 (all three tag variations). Error, ± SD.

    Techniques Used: Activity Assay, Expressing, Sedimentation, SDS-Gel, Staining, In Vitro

    10) Product Images from "H2O2 dynamics in the malaria parasite Plasmodium falciparum"

    Article Title: H2O2 dynamics in the malaria parasite Plasmodium falciparum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174837

    Dynamic range of roGFP2-Orp1 and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM DTT at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p
    Figure Legend Snippet: Dynamic range of roGFP2-Orp1 and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM DTT at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p

    Techniques Used: Transfection, Lysis, Confocal Laser Scanning Microscopy, Fluorescence, Incubation

    11) Product Images from "A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ domain interactions"

    Article Title: A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ domain interactions

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1633250100

    Oligomeric status and the regulation of CFTR channel activity by GST-PDZ1 and GST-PDZ2. ( A ) Analysis of chemically cross-linked proteins under nonreducing (-DTT) and reducing (+DTT) conditions by SDS/PAGE. In the absence of DTT ( Left ), the top bands (double arrowheads) represent cross-linked dimers of GST-PDZ1 and GST-PDZ2, and the (faint) bottom band (single arrowhead) represents uncross-linked monomer. In contrast, no bands corresponding to dimers of individual PDZ domains are seen, suggesting that they exist as monomers. The dimers migrate as doublets due to the presence of intramolecular cross-links. In the presence of DTT ( Right ), only monomeric species are seen. ( B ) Functional effects of GST-PDZ1 and GST-PDZ2 on CFTR channel activity. Representative experiment containing four CFTR channels and their stimulation by addition of GST-PDZ1 and effect of subsequent addition of PKC on CFTR P o . Numbers indicate number of open channel levels. Summary of CFTR P o under various experimental conditions using GST-PDZ1 ( C ) and GST-PDZ2 ( D ). * and ** indicate significant difference of P o after addition of PDZ1–2 peptide and after addition of PKC, respectively.
    Figure Legend Snippet: Oligomeric status and the regulation of CFTR channel activity by GST-PDZ1 and GST-PDZ2. ( A ) Analysis of chemically cross-linked proteins under nonreducing (-DTT) and reducing (+DTT) conditions by SDS/PAGE. In the absence of DTT ( Left ), the top bands (double arrowheads) represent cross-linked dimers of GST-PDZ1 and GST-PDZ2, and the (faint) bottom band (single arrowhead) represents uncross-linked monomer. In contrast, no bands corresponding to dimers of individual PDZ domains are seen, suggesting that they exist as monomers. The dimers migrate as doublets due to the presence of intramolecular cross-links. In the presence of DTT ( Right ), only monomeric species are seen. ( B ) Functional effects of GST-PDZ1 and GST-PDZ2 on CFTR channel activity. Representative experiment containing four CFTR channels and their stimulation by addition of GST-PDZ1 and effect of subsequent addition of PKC on CFTR P o . Numbers indicate number of open channel levels. Summary of CFTR P o under various experimental conditions using GST-PDZ1 ( C ) and GST-PDZ2 ( D ). * and ** indicate significant difference of P o after addition of PDZ1–2 peptide and after addition of PKC, respectively.

    Techniques Used: Activity Assay, SDS Page, Functional Assay

    12) Product Images from "Fluorescent-responsive Synthetic C1b Domains of Protein Kinase C? as Reporters of Specific High Affinity Ligand Binding"

    Article Title: Fluorescent-responsive Synthetic C1b Domains of Protein Kinase C? as Reporters of Specific High Affinity Ligand Binding

    Journal: Bioconjugate chemistry

    doi: 10.1021/bc100414a

    Schematic representation of construction of dansyl-labaled δC1b. i) 100 mM phosphate buffer (pH 8.5), 6 M Gn·HCl, 2 mM EDTA, TCEP·HCl, thiophenol, N 2 , 37 °C; ii) 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1mM DTT, 0.1
    Figure Legend Snippet: Schematic representation of construction of dansyl-labaled δC1b. i) 100 mM phosphate buffer (pH 8.5), 6 M Gn·HCl, 2 mM EDTA, TCEP·HCl, thiophenol, N 2 , 37 °C; ii) 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1mM DTT, 0.1

    Techniques Used:

    13) Product Images from "Reversal of paclitaxel resistance in human ovarian cancer cells with redox-responsive micelles consisting of α-tocopheryl succinate-based polyphosphoester copolymers"

    Article Title: Reversal of paclitaxel resistance in human ovarian cancer cells with redox-responsive micelles consisting of α-tocopheryl succinate-based polyphosphoester copolymers

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2016.150

    The chemical structure and 1 H NMR spectrum of PSST polymer are shown in (A). The gel permeation chromatography (GPC) curves of PSST polymer treated with or without 10 mmol/L DTT for 6 h were determined (B). The critical micellar concentration (CMC) value of PSST polymer was determined by the change of pyrene fluorescence (C). The MTT assay was used to examine viability of A2780 and A2780/PTX cells treated with PSST polymer for 48 h (D).
    Figure Legend Snippet: The chemical structure and 1 H NMR spectrum of PSST polymer are shown in (A). The gel permeation chromatography (GPC) curves of PSST polymer treated with or without 10 mmol/L DTT for 6 h were determined (B). The critical micellar concentration (CMC) value of PSST polymer was determined by the change of pyrene fluorescence (C). The MTT assay was used to examine viability of A2780 and A2780/PTX cells treated with PSST polymer for 48 h (D).

    Techniques Used: Nuclear Magnetic Resonance, GPC Assay, Gel Permeation Chromatography, Concentration Assay, Fluorescence, MTT Assay

    14) Product Images from "Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli"

    Article Title: Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

    Journal: Current protocols in protein science / editorial board, John E. Coligan ... [et al.]

    doi: 10.1002/0471140864.ps0603s38

    Gel filtration of an extract containing HIV-1 protease, using Superdex 200 in 4 M guanidine·HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris·Cl (pH 7.5)/4 mM guanidine·Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr). The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) was further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS-PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).
    Figure Legend Snippet: Gel filtration of an extract containing HIV-1 protease, using Superdex 200 in 4 M guanidine·HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris·Cl (pH 7.5)/4 mM guanidine·Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr). The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) was further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS-PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).

    Techniques Used: Filtration, Flow Cytometry, Purification, SDS Page, Migration

    15) Product Images from "Real-Time Imaging of the Intracellular Glutathione Redox Potential in the Malaria Parasite Plasmodium falciparum"

    Article Title: Real-Time Imaging of the Intracellular Glutathione Redox Potential in the Malaria Parasite Plasmodium falciparum

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003782

    Real-time imaging of the glutathione redox potential in P. falciparum . A. Confocal live cell images of 3D7 and Dd2 parasites showing the expression of hGrx1-roGFP2 localized in the cytosol. B . hGrx1-roGFP2 is a fusion protein with human glutaredoxin 1 (hGrx1, black) fused to the N-terminal end of roGFP2 (green) through a linker (red) comprising 30 amino acids (Gly-Gly-Ser-Gly-Gly) 6 . Live cell imaging of the trophozoite stages of ( C ) 3D7 hGrx1-roGFP2 and ( D ) Dd2 hGrx1-roGFP2 . After 60 s, 3D7 ( C ) and Dd2 ( D ) parasites were treated with 1 mM diamide followed 4 min later by addition of 10 mM dithiothreitol (DTT). 405 nm, 488 nm, merge (405/488 nm), and false color ratio images at different time points are shown. E . The ratio of emissions (ratio 405/488 nm) after excitation at 405 and 488 nm was computed for both strains and plotted against time. Data from 5 trophozoites for each strain were analyzed per data point. F . Fluorescence ratio as a function of time. The ratio 405/488 nm remained stable over a period of 10 min. Data from 5 trophozoites for each strain were analyzed per data point. Mean and standard errors of the mean are shown.
    Figure Legend Snippet: Real-time imaging of the glutathione redox potential in P. falciparum . A. Confocal live cell images of 3D7 and Dd2 parasites showing the expression of hGrx1-roGFP2 localized in the cytosol. B . hGrx1-roGFP2 is a fusion protein with human glutaredoxin 1 (hGrx1, black) fused to the N-terminal end of roGFP2 (green) through a linker (red) comprising 30 amino acids (Gly-Gly-Ser-Gly-Gly) 6 . Live cell imaging of the trophozoite stages of ( C ) 3D7 hGrx1-roGFP2 and ( D ) Dd2 hGrx1-roGFP2 . After 60 s, 3D7 ( C ) and Dd2 ( D ) parasites were treated with 1 mM diamide followed 4 min later by addition of 10 mM dithiothreitol (DTT). 405 nm, 488 nm, merge (405/488 nm), and false color ratio images at different time points are shown. E . The ratio of emissions (ratio 405/488 nm) after excitation at 405 and 488 nm was computed for both strains and plotted against time. Data from 5 trophozoites for each strain were analyzed per data point. F . Fluorescence ratio as a function of time. The ratio 405/488 nm remained stable over a period of 10 min. Data from 5 trophozoites for each strain were analyzed per data point. Mean and standard errors of the mean are shown.

    Techniques Used: Imaging, Expressing, Live Cell Imaging, Fluorescence

    16) Product Images from "A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ domain interactions"

    Article Title: A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ domain interactions

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1633250100

    Oligomeric status and the regulation of CFTR channel activity by GST-PDZ1 and GST-PDZ2. ( A ) Analysis of chemically cross-linked proteins under nonreducing (-DTT) and reducing (+DTT) conditions by SDS/PAGE. In the absence of DTT ( Left ), the top bands (double arrowheads) represent cross-linked dimers of GST-PDZ1 and GST-PDZ2, and the (faint) bottom band (single arrowhead) represents uncross-linked monomer. In contrast, no bands corresponding to dimers of individual PDZ domains are seen, suggesting that they exist as monomers. The dimers migrate as doublets due to the presence of intramolecular cross-links. In the presence of DTT ( Right ), only monomeric species are seen. ( B ) Functional effects of GST-PDZ1 and GST-PDZ2 on CFTR channel activity. Representative experiment containing four CFTR channels and their stimulation by addition of GST-PDZ1 and effect of subsequent addition of PKC on CFTR P o . Numbers indicate number of open channel levels. Summary of CFTR P o under various experimental conditions using GST-PDZ1 ( C ) and GST-PDZ2 ( D ). * and ** indicate significant difference of P o after addition of PDZ1–2 peptide and after addition of PKC, respectively.
    Figure Legend Snippet: Oligomeric status and the regulation of CFTR channel activity by GST-PDZ1 and GST-PDZ2. ( A ) Analysis of chemically cross-linked proteins under nonreducing (-DTT) and reducing (+DTT) conditions by SDS/PAGE. In the absence of DTT ( Left ), the top bands (double arrowheads) represent cross-linked dimers of GST-PDZ1 and GST-PDZ2, and the (faint) bottom band (single arrowhead) represents uncross-linked monomer. In contrast, no bands corresponding to dimers of individual PDZ domains are seen, suggesting that they exist as monomers. The dimers migrate as doublets due to the presence of intramolecular cross-links. In the presence of DTT ( Right ), only monomeric species are seen. ( B ) Functional effects of GST-PDZ1 and GST-PDZ2 on CFTR channel activity. Representative experiment containing four CFTR channels and their stimulation by addition of GST-PDZ1 and effect of subsequent addition of PKC on CFTR P o . Numbers indicate number of open channel levels. Summary of CFTR P o under various experimental conditions using GST-PDZ1 ( C ) and GST-PDZ2 ( D ). * and ** indicate significant difference of P o after addition of PDZ1–2 peptide and after addition of PKC, respectively.

    Techniques Used: Activity Assay, SDS Page, Functional Assay

    17) Product Images from "Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis during Infection"

    Article Title: Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis during Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003902

    Mrx1 catalyzes specific equilibration between mycothiol redox system and roGFP2 in vitro and in vivo . (A) Pre-reduced roGFP2 (lane 1), Mrx1-roGFP2 (lane 2), Mrx1(AGYC)-roGFP2 (lane 3), and Mrx1(CGYA)-roGFP2 (lane 4) were exposed to 50 µM of MSSM for 10 min and ratiometric sensor response was measured. (B) Pre-reduced Mrx1-roGFP2 was treated with 1 µM of MSSM, GSSG, cystine (Cys 2 ) or 2-hydroxyethyl disulfide (HED) and ratiometric sensor response was measured at various time points. (C) Molecular mechanism showing the reduction of oxidized Mrx1-roGFP2 by MSH/Mtr/NADPH pathway. (D) Oxidized Mrx1-roGFP2 was added as a substrate to the MSH/Mtr/NADPH redox pathway and ratiometric sensor response was measured over time. A control reaction in the absence of MSH was performed in parallel. (E) Reduction of oxidized roGFP2 (lane 1), Mrx1-roGFP2 (lane 2), Mrx1(AGYC)-roGFP2 (lane 3), and Mrx1(CGYA)-roGFP2 (lane 4) by MSH/Mtr/NADPH redox pathway. Maximum ratio change after 150 min of incubation with MSH/Mtr/NADPH reaction mixture is shown. (F) Mrx1-roGFP2 is extremely sensitive towards small changes in OxD MSH . Reduced uncoupled roGFP2 and Mrx1-roGFP2 proteins were incubated with mycothiol solutions (1 mM total) containing increasing fractions of MSSM for a maximum of 30 sec and ratiometric sensor response was measured. Note that the response of Mrx1-roGFP2 becomes exceedingly linear in the window between 10% to 90% oxidation, suggesting that the biosensor can effectively measure changes in E MSH within this range of probe oxidation. (G) Excitation spectra of Msm expressing Mrx1-roGFP2 upon treatment with 0.4 mM of diamide (oxidant) or 10 mM of DTT (reductant) for 5 min. (H) Msm expressing Mrx1-roGFP2 was either left untreated (control) or exposed to 50 µM dequalinium, cisplatin and 5-methoxyindole-2-carboxylic acid (MICA) and ratiometric sensor response was measured after 2 h and 24 h post-exposure. p-values shown in the panel were calculated by comparing untreated group and dequalinium treated group. (I) Percentage of OxD Mrx1-roGFP2 in exponentially grown Msm , MsmΔmshA , MsmΔmshD , and MsmΔsigH was calculated (see Materials and Methods for mathematical definition). Note that biosensor is completely oxidized (∼95%) in the absence of MSH reducing system in MsmΔmshA . p-values shown in the panel were calculated by independently comparing MsmΔmshA and MsmΔmshD groups with the Msm group. Error bars represent standard deviations from the mean. * p
    Figure Legend Snippet: Mrx1 catalyzes specific equilibration between mycothiol redox system and roGFP2 in vitro and in vivo . (A) Pre-reduced roGFP2 (lane 1), Mrx1-roGFP2 (lane 2), Mrx1(AGYC)-roGFP2 (lane 3), and Mrx1(CGYA)-roGFP2 (lane 4) were exposed to 50 µM of MSSM for 10 min and ratiometric sensor response was measured. (B) Pre-reduced Mrx1-roGFP2 was treated with 1 µM of MSSM, GSSG, cystine (Cys 2 ) or 2-hydroxyethyl disulfide (HED) and ratiometric sensor response was measured at various time points. (C) Molecular mechanism showing the reduction of oxidized Mrx1-roGFP2 by MSH/Mtr/NADPH pathway. (D) Oxidized Mrx1-roGFP2 was added as a substrate to the MSH/Mtr/NADPH redox pathway and ratiometric sensor response was measured over time. A control reaction in the absence of MSH was performed in parallel. (E) Reduction of oxidized roGFP2 (lane 1), Mrx1-roGFP2 (lane 2), Mrx1(AGYC)-roGFP2 (lane 3), and Mrx1(CGYA)-roGFP2 (lane 4) by MSH/Mtr/NADPH redox pathway. Maximum ratio change after 150 min of incubation with MSH/Mtr/NADPH reaction mixture is shown. (F) Mrx1-roGFP2 is extremely sensitive towards small changes in OxD MSH . Reduced uncoupled roGFP2 and Mrx1-roGFP2 proteins were incubated with mycothiol solutions (1 mM total) containing increasing fractions of MSSM for a maximum of 30 sec and ratiometric sensor response was measured. Note that the response of Mrx1-roGFP2 becomes exceedingly linear in the window between 10% to 90% oxidation, suggesting that the biosensor can effectively measure changes in E MSH within this range of probe oxidation. (G) Excitation spectra of Msm expressing Mrx1-roGFP2 upon treatment with 0.4 mM of diamide (oxidant) or 10 mM of DTT (reductant) for 5 min. (H) Msm expressing Mrx1-roGFP2 was either left untreated (control) or exposed to 50 µM dequalinium, cisplatin and 5-methoxyindole-2-carboxylic acid (MICA) and ratiometric sensor response was measured after 2 h and 24 h post-exposure. p-values shown in the panel were calculated by comparing untreated group and dequalinium treated group. (I) Percentage of OxD Mrx1-roGFP2 in exponentially grown Msm , MsmΔmshA , MsmΔmshD , and MsmΔsigH was calculated (see Materials and Methods for mathematical definition). Note that biosensor is completely oxidized (∼95%) in the absence of MSH reducing system in MsmΔmshA . p-values shown in the panel were calculated by independently comparing MsmΔmshA and MsmΔmshD groups with the Msm group. Error bars represent standard deviations from the mean. * p

    Techniques Used: In Vitro, In Vivo, Incubation, Size-exclusion Chromatography, Expressing

    Emergence of redox heterogeneity within Mtb population inside macrophages. PMA-differentiated THP-1 cells were infected with Mtb H37Rv expressing Mrx1-roGFP2 (moi: 10) and ∼30,000 cells were analyzed by flow cytometry by exciting at 405 and 488 nm lasers at a constant emission (510 nm). The program BD FACS suite software was used to analyze the population distribution of Mtb , and each population was represented by an unique color. Using automatic and manual gating options, a strategy was adopted to categorize Mtb population into three subpopulations: E MSH -oxidized, E MSH -reduced and E MSH -basal. Number of events per subpopulation was counted and representative percentage of each subpopulation was estimated. Gates were selected on the basis of complete oxidation by 1 mM CHP ( E MSH -oxidized) and complete reduction by 10 mM DTT ( E MSH -reduced). Dot plots show shift in population towards oxidizing or reducing after treatment with (A) CHP and (B) DTT, respectively. (C) Overlay spectra of dot plots derived from CHP and DTT treatment of infected THP-1 cells. (D) Bar graph represents the ratiometric sensor response. (E) Dot plot of THP-1 cells infected with H37Rv expressing Mrx1-roGFP2 at 72 h p.i. (F) Bar graph represents percentage of bacilli in each subpopulation. (G) Dot plot of H37Rv expressing Mrx1-roGFP2 grown in 7H9 medium. (H) Bar graph represents percentage of bacilli in each subpopulation. (I) THP-1 cells were infected with BCG expressing Mrx1-roGFP2 and ∼30,000 cells were analyzed by flow cytometry at 24 h p.i. (J) In a parallel set, BCG infected cells were first fixed by NEM and PFA followed by flow cytometry. (K) Shown is the dot plot of BCG infected THP-1 cells with and without NEM-PFA treatment. Note that redox heterogeneity was preserved independent of NEM-PFA treatment. Bar graph represents percentage of bacilli in each subpopulation. The E MSH of mycobacterial cells in vitro and inside macrophages was calculated by fitting Mrx1-roGFP2 ratios into the in vitro redox calibration curve (see SI Materials and Methods ). Color codes representing each subpopulation with a defined average E MSH in the panels are shown at the bottom of the figure. Error bars represent standard deviations from the mean of at least three independent experiments.
    Figure Legend Snippet: Emergence of redox heterogeneity within Mtb population inside macrophages. PMA-differentiated THP-1 cells were infected with Mtb H37Rv expressing Mrx1-roGFP2 (moi: 10) and ∼30,000 cells were analyzed by flow cytometry by exciting at 405 and 488 nm lasers at a constant emission (510 nm). The program BD FACS suite software was used to analyze the population distribution of Mtb , and each population was represented by an unique color. Using automatic and manual gating options, a strategy was adopted to categorize Mtb population into three subpopulations: E MSH -oxidized, E MSH -reduced and E MSH -basal. Number of events per subpopulation was counted and representative percentage of each subpopulation was estimated. Gates were selected on the basis of complete oxidation by 1 mM CHP ( E MSH -oxidized) and complete reduction by 10 mM DTT ( E MSH -reduced). Dot plots show shift in population towards oxidizing or reducing after treatment with (A) CHP and (B) DTT, respectively. (C) Overlay spectra of dot plots derived from CHP and DTT treatment of infected THP-1 cells. (D) Bar graph represents the ratiometric sensor response. (E) Dot plot of THP-1 cells infected with H37Rv expressing Mrx1-roGFP2 at 72 h p.i. (F) Bar graph represents percentage of bacilli in each subpopulation. (G) Dot plot of H37Rv expressing Mrx1-roGFP2 grown in 7H9 medium. (H) Bar graph represents percentage of bacilli in each subpopulation. (I) THP-1 cells were infected with BCG expressing Mrx1-roGFP2 and ∼30,000 cells were analyzed by flow cytometry at 24 h p.i. (J) In a parallel set, BCG infected cells were first fixed by NEM and PFA followed by flow cytometry. (K) Shown is the dot plot of BCG infected THP-1 cells with and without NEM-PFA treatment. Note that redox heterogeneity was preserved independent of NEM-PFA treatment. Bar graph represents percentage of bacilli in each subpopulation. The E MSH of mycobacterial cells in vitro and inside macrophages was calculated by fitting Mrx1-roGFP2 ratios into the in vitro redox calibration curve (see SI Materials and Methods ). Color codes representing each subpopulation with a defined average E MSH in the panels are shown at the bottom of the figure. Error bars represent standard deviations from the mean of at least three independent experiments.

    Techniques Used: Infection, Expressing, Flow Cytometry, Cytometry, FACS, Software, Derivative Assay, In Vitro

    18) Product Images from "Network approach identifies Pacer as an autophagy protein involved in ALS pathogenesis"

    Article Title: Network approach identifies Pacer as an autophagy protein involved in ALS pathogenesis

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-019-0313-9

    Pacer levels are reduced during ALS pathology. a Human Pacer (hPacer) and human Rubicon (hRubicon) protein levels were determined in post-mortem spinal cord sections from sALS patients and age-matched control subjects. Left panel, cervical spinal cord section with Controls n = 2 and sALS patients n = 6; middle panel, thoracic spinal cord section with Controls n = 2 and sALS patients n = 7; and right panel, lumbar spinal cord section with Controls n = 6 and sALS patients n = 7. β-Actin serves as a loading control. Densitometric quantifications of hPacer and hRubicon normalized to β-Actin levels are shown. b Pacer and Rubicon protein levels were determined in lumbar spinal cord samples of late symptomatic TDP43 A315T transgenic mice (TDP43 A315T -Tg, n = 5) and their non-transgenic littermate controls ( n = 3), respectively. TDP43 aggregate levels under non-reducing (−DTT) conditions are shown as positive controls. β-Actin serves as a loading control. Densitometric quantifications of Pacer protein levels normalized to β-Actin levels are shown. c , Pacer and Rubicon protein levels were determined in the lumbar spinal cord of late symptomatic SOD1 G93A transgenic mice (SOD1 G93A -Tg) and their non-transgenic littermate controls (both groups n = 7). p62 protein levels were detected as a positive control of impaired autophagy. SOD1 aggregate levels under non-reduced (−DTT) conditions are shown as a positive control for SOD1 G93A -Tg mice. β-Actin serves as a loading control. Densitometric quantifications of Pacer protein levels normalized to β-Actin levels are shown. In a - c Statistical analyses were performed using Student’s t-test. Mean and SEM with only statistical significant p-values are shown: *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001
    Figure Legend Snippet: Pacer levels are reduced during ALS pathology. a Human Pacer (hPacer) and human Rubicon (hRubicon) protein levels were determined in post-mortem spinal cord sections from sALS patients and age-matched control subjects. Left panel, cervical spinal cord section with Controls n = 2 and sALS patients n = 6; middle panel, thoracic spinal cord section with Controls n = 2 and sALS patients n = 7; and right panel, lumbar spinal cord section with Controls n = 6 and sALS patients n = 7. β-Actin serves as a loading control. Densitometric quantifications of hPacer and hRubicon normalized to β-Actin levels are shown. b Pacer and Rubicon protein levels were determined in lumbar spinal cord samples of late symptomatic TDP43 A315T transgenic mice (TDP43 A315T -Tg, n = 5) and their non-transgenic littermate controls ( n = 3), respectively. TDP43 aggregate levels under non-reducing (−DTT) conditions are shown as positive controls. β-Actin serves as a loading control. Densitometric quantifications of Pacer protein levels normalized to β-Actin levels are shown. c , Pacer and Rubicon protein levels were determined in the lumbar spinal cord of late symptomatic SOD1 G93A transgenic mice (SOD1 G93A -Tg) and their non-transgenic littermate controls (both groups n = 7). p62 protein levels were detected as a positive control of impaired autophagy. SOD1 aggregate levels under non-reduced (−DTT) conditions are shown as a positive control for SOD1 G93A -Tg mice. β-Actin serves as a loading control. Densitometric quantifications of Pacer protein levels normalized to β-Actin levels are shown. In a - c Statistical analyses were performed using Student’s t-test. Mean and SEM with only statistical significant p-values are shown: *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001

    Techniques Used: Transgenic Assay, Mouse Assay, Positive Control

    Depletion of Pacer impairs autophagosome formation and promotes SOD1 aggregation. a Autophagy flux under Pacer knockdown. Cells were treated with EBSS medium or/and lysosome inhibitors (Lys. Inh.) for 0.5, 2 and 4 h. Cell extracts were subjected to Western blot. As a mock control, a scrambled shRNA (shCtrl) construct was used. Pacer, Beclin1, p62 and LC3-II formation levels were determined. β-Actin serves as a loading control . b Densitometric quantifications of LC3-II flux (n = 3). One-way ANOVA and Bonferroni’s post hoc tests were performed.Mean and SEM with only statistically significant p-values are shown: *, p ≤ 0.05. c-f , NSC34 cells depleted of Pacer were transiently co-transfected with expression vectors for human wild-type or mutant SOD1 G93A fused to EGFP. When indicated, human Pacer (hPacer-V5) was co-expressed. c and d , after 48 h, SOD1 aggregation was assessed under non-reducing (−DTT) conditions. Cell extracts were prepared in 1% Triton X-100 buffer or 1% SDS buffer for Western blot and filter trap assays, respectively. In c HSP90 serves as a loading control. e SOD1 inclusions in NSC34 cells were assessed by confocal microscopy. Percentages of cells with SOD1 WT -EGFP or SOD1 G93A -EGFP inclusions are shown. f Percentage of cell death was quantified at 72 h (SytoxBlue positive, SB+) in NSC34 stable lines expressing shPacer or shCtrl transiently transfected with EGFP, SOD1 WT or SOD1 G93A , and hPacer-V5. In e and f statistical analyses were performed using one-way ANOVA and Bonferroni’s post-hoc tests. Mean and SEM with only statistically significant p-values are shown: *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001
    Figure Legend Snippet: Depletion of Pacer impairs autophagosome formation and promotes SOD1 aggregation. a Autophagy flux under Pacer knockdown. Cells were treated with EBSS medium or/and lysosome inhibitors (Lys. Inh.) for 0.5, 2 and 4 h. Cell extracts were subjected to Western blot. As a mock control, a scrambled shRNA (shCtrl) construct was used. Pacer, Beclin1, p62 and LC3-II formation levels were determined. β-Actin serves as a loading control . b Densitometric quantifications of LC3-II flux (n = 3). One-way ANOVA and Bonferroni’s post hoc tests were performed.Mean and SEM with only statistically significant p-values are shown: *, p ≤ 0.05. c-f , NSC34 cells depleted of Pacer were transiently co-transfected with expression vectors for human wild-type or mutant SOD1 G93A fused to EGFP. When indicated, human Pacer (hPacer-V5) was co-expressed. c and d , after 48 h, SOD1 aggregation was assessed under non-reducing (−DTT) conditions. Cell extracts were prepared in 1% Triton X-100 buffer or 1% SDS buffer for Western blot and filter trap assays, respectively. In c HSP90 serves as a loading control. e SOD1 inclusions in NSC34 cells were assessed by confocal microscopy. Percentages of cells with SOD1 WT -EGFP or SOD1 G93A -EGFP inclusions are shown. f Percentage of cell death was quantified at 72 h (SytoxBlue positive, SB+) in NSC34 stable lines expressing shPacer or shCtrl transiently transfected with EGFP, SOD1 WT or SOD1 G93A , and hPacer-V5. In e and f statistical analyses were performed using one-way ANOVA and Bonferroni’s post-hoc tests. Mean and SEM with only statistically significant p-values are shown: *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001

    Techniques Used: Western Blot, shRNA, Construct, Transfection, Expressing, Mutagenesis, Confocal Microscopy

    19) Product Images from "Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway"

    Article Title: Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

    Journal: Redox Biology

    doi: 10.1016/j.redox.2019.101400

    BQ-induced enzyme inactivation and covalent modification of purified Trx1 protein. (A) Concentration-dependent inhibition of Trx1 activity by BQ. DTT-reduced Trx1 (2.5 μM) was incubated with BQ (0.5–10 μM) in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), with Trx1 activity determined after 5 min of treatment. (B) BQ-induced quinoprotein detection. DTT-reduced Trx1 (10 μM) was incubated with BQ (5–40 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed by SDS-PAGE followed by NBT redox staining. (C) Free thiol detection using BEI labelling. DTT-reduced Trx1 (5 μM) was incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by incubation with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection. * p
    Figure Legend Snippet: BQ-induced enzyme inactivation and covalent modification of purified Trx1 protein. (A) Concentration-dependent inhibition of Trx1 activity by BQ. DTT-reduced Trx1 (2.5 μM) was incubated with BQ (0.5–10 μM) in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), with Trx1 activity determined after 5 min of treatment. (B) BQ-induced quinoprotein detection. DTT-reduced Trx1 (10 μM) was incubated with BQ (5–40 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed by SDS-PAGE followed by NBT redox staining. (C) Free thiol detection using BEI labelling. DTT-reduced Trx1 (5 μM) was incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by incubation with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection. * p

    Techniques Used: Modification, Purification, Concentration Assay, Inhibition, Activity Assay, Incubation, SDS Page, Staining

    Identification of BQ-modified Cys residues in Trx1. DTT-reduced Trx1 (10 μM) was incubated with BQ (50 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The BQ-treated Trx1 was then digested using Lys-C at 37 °C overnight and analysed by LC-MS/MS (seen Materials and methods for further details). (A) Peptide 1, (B) Peptide 2, (C) Peptide 3 (cf. Table 1 ).
    Figure Legend Snippet: Identification of BQ-modified Cys residues in Trx1. DTT-reduced Trx1 (10 μM) was incubated with BQ (50 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The BQ-treated Trx1 was then digested using Lys-C at 37 °C overnight and analysed by LC-MS/MS (seen Materials and methods for further details). (A) Peptide 1, (B) Peptide 2, (C) Peptide 3 (cf. Table 1 ).

    Techniques Used: Modification, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Effect of GSH on BQ-induced enzyme activity loss, protein cross-linking and modification. Panels A, C, E: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) in the presence of GSH (5–1000 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The samples were split into three portions. The remaining Trx activity was measured (panel A), samples were analysed by SDS-PAGE followed by silver staining (panel C), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel E). Panels B, D, F: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), then GSH (5–1000 μM) was added and incubated for another 5 min. The samples were then split into three portions and analysed, as described above, for remaining Trx activity (panel B), by SDS-PAGE followed by silver staining (panel D), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel F). Data (mean ± standard deviations) from 3 independent experiments are presented in panels A and B. Representative images from 3 independent experiments are shown in panels (C - F). * p
    Figure Legend Snippet: Effect of GSH on BQ-induced enzyme activity loss, protein cross-linking and modification. Panels A, C, E: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) in the presence of GSH (5–1000 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The samples were split into three portions. The remaining Trx activity was measured (panel A), samples were analysed by SDS-PAGE followed by silver staining (panel C), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel E). Panels B, D, F: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), then GSH (5–1000 μM) was added and incubated for another 5 min. The samples were then split into three portions and analysed, as described above, for remaining Trx activity (panel B), by SDS-PAGE followed by silver staining (panel D), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel F). Data (mean ± standard deviations) from 3 independent experiments are presented in panels A and B. Representative images from 3 independent experiments are shown in panels (C - F). * p

    Techniques Used: Activity Assay, Modification, Incubation, SDS Page, Silver Staining

    BQ-induced protein cross-linking. Human Trx1 (5 μM, A and B) and E. coli Trx (5 μM, C and D), pre-treated with DTT, were incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed under non-reducing (A and C) and reducing (B and D) conditions, and then analysed by SDS-PAGE with silver staining. Representative images from 3 independent experiments are shown in each panel.
    Figure Legend Snippet: BQ-induced protein cross-linking. Human Trx1 (5 μM, A and B) and E. coli Trx (5 μM, C and D), pre-treated with DTT, were incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed under non-reducing (A and C) and reducing (B and D) conditions, and then analysed by SDS-PAGE with silver staining. Representative images from 3 independent experiments are shown in each panel.

    Techniques Used: Incubation, SDS Page, Silver Staining

    20) Product Images from "Insulin Capture by an Insulin-linked Polymorphic Region G-quadruplex DNA Oligonucleotide"

    Article Title: Insulin Capture by an Insulin-linked Polymorphic Region G-quadruplex DNA Oligonucleotide

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja056097c

    MALDI mass spectra of collections from affinity capillary chromatography capture experiment of 500 μM insulin incubated for 30 min with 500 μM DTT before loading onto ILPR 2 capillary.
    Figure Legend Snippet: MALDI mass spectra of collections from affinity capillary chromatography capture experiment of 500 μM insulin incubated for 30 min with 500 μM DTT before loading onto ILPR 2 capillary.

    Techniques Used: Chromatography, Incubation

    21) Product Images from "Synthetic substrates for measuring activity of autophagy proteases"

    Article Title: Synthetic substrates for measuring activity of autophagy proteases

    Journal: Autophagy

    doi: 10.4161/auto.6.7.13075

    Characterization of Atg4B activity using synthetic peptide substrates. (A) Various concentrations of recombinant Atg4B were incubated with 100 µM synthetic tetrapeptides conjugated with AFC in 50 mM Tris-HCl, pH 8.0, 5 mM DTT at 37°C for
    Figure Legend Snippet: Characterization of Atg4B activity using synthetic peptide substrates. (A) Various concentrations of recombinant Atg4B were incubated with 100 µM synthetic tetrapeptides conjugated with AFC in 50 mM Tris-HCl, pH 8.0, 5 mM DTT at 37°C for

    Techniques Used: Activity Assay, Recombinant, Incubation

    22) Product Images from "Microscale Bioadhesive Hydrogel Arrays for Cell Engineering Applications"

    Article Title: Microscale Bioadhesive Hydrogel Arrays for Cell Engineering Applications

    Journal: Cellular and molecular bioengineering

    doi: 10.1007/s12195-014-0353-8

    Bio-adhesive, cell encapsulated IPN of PEG-MAL and gelatin-silicate nanoparticles (NP). (a) Schematic of bio-adhesive cell supportive microenvironment consisting of 4-arm PEG-MAL crosslinked with DTT and coated with a stable IPN of gelatin with silicate
    Figure Legend Snippet: Bio-adhesive, cell encapsulated IPN of PEG-MAL and gelatin-silicate nanoparticles (NP). (a) Schematic of bio-adhesive cell supportive microenvironment consisting of 4-arm PEG-MAL crosslinked with DTT and coated with a stable IPN of gelatin with silicate

    Techniques Used:

    23) Product Images from "Characterization of Polyethylene Glycol–Reinforced Alginate Microcapsules for Mechanically Stable Cell Immunoisolation"

    Article Title: Characterization of Polyethylene Glycol–Reinforced Alginate Microcapsules for Mechanically Stable Cell Immunoisolation

    Journal: Macromolecular materials and engineering

    doi: 10.1002/mame.201800679

    Microcapsule Permeability Analysis and Extrapolated Diffusion Coefficients. A–E) 4 and 10 kDa FITC-Dextran release curves for the ALG, MM-DTT, MM-PEGSH, PEG-DTT, and PEG-PEGSH capsules, respectively. FITC diffusion into solution was sampled periodically over time from t = 0 h to t = 24 h. F) Theoretical diffusion coefficients for the materials were extrapolated from the experimental data.
    Figure Legend Snippet: Microcapsule Permeability Analysis and Extrapolated Diffusion Coefficients. A–E) 4 and 10 kDa FITC-Dextran release curves for the ALG, MM-DTT, MM-PEGSH, PEG-DTT, and PEG-PEGSH capsules, respectively. FITC diffusion into solution was sampled periodically over time from t = 0 h to t = 24 h. F) Theoretical diffusion coefficients for the materials were extrapolated from the experimental data.

    Techniques Used: Permeability, Diffusion-based Assay

    24) Product Images from "Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus"

    Article Title: Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus

    Journal: Biochemical Journal

    doi: 10.1042/BJ20140463

    Gun4 is able to associate with and stimulate activity of ΔN159ChlH ( A ) FRET association experiment between Gun4 and WT ChlH (●) or ΔN159ChlH (○). Assays contained 0.1 μM ChlH, in 50 mM Tricine/NaOH, 0.3 M glycerol and 200 mM NaCl, pH 7.9, at 45°C. ChlH was labelled with Atto 488 and Gun4 labelled with Alexa Fluor 532. The quenching of emission of Atto 488 was monitored at 524 nm. A.U.–arbitrary units. The curves can be described by eqn ( 1 ) with the concentration term, [ChlH], held at 0.1 μM with characterizing parameters: WT ChlH K d =0.009±0.003 μM and Δ159ChlH K d =0.016±0.004 μM. ( B ) Assembly titrations of MgCH between Gun4. Assays contained 0.1 μM ChlD, 0.2 μM ChlI, 0.4 μM ChlH in 50 mM Mops/KOH, 0.3 M glycerol, 1 mM DTT, 10 mM free Mg 2+ , I =0.1 with KCl and 8 μM D IX , pH 7.7, at 45°C. The curves can be described by eqn ( 2 ). n d was held at 0.4 (i.e. one binding site on ChlH for Gun4) based on the fit in ( A ) with characterizing parameters WT ChlH K app =0.39±0.14 μM and Δ159ChlH K app =0.05±0.03 μM.
    Figure Legend Snippet: Gun4 is able to associate with and stimulate activity of ΔN159ChlH ( A ) FRET association experiment between Gun4 and WT ChlH (●) or ΔN159ChlH (○). Assays contained 0.1 μM ChlH, in 50 mM Tricine/NaOH, 0.3 M glycerol and 200 mM NaCl, pH 7.9, at 45°C. ChlH was labelled with Atto 488 and Gun4 labelled with Alexa Fluor 532. The quenching of emission of Atto 488 was monitored at 524 nm. A.U.–arbitrary units. The curves can be described by eqn ( 1 ) with the concentration term, [ChlH], held at 0.1 μM with characterizing parameters: WT ChlH K d =0.009±0.003 μM and Δ159ChlH K d =0.016±0.004 μM. ( B ) Assembly titrations of MgCH between Gun4. Assays contained 0.1 μM ChlD, 0.2 μM ChlI, 0.4 μM ChlH in 50 mM Mops/KOH, 0.3 M glycerol, 1 mM DTT, 10 mM free Mg 2+ , I =0.1 with KCl and 8 μM D IX , pH 7.7, at 45°C. The curves can be described by eqn ( 2 ). n d was held at 0.4 (i.e. one binding site on ChlH for Gun4) based on the fit in ( A ) with characterizing parameters WT ChlH K app =0.39±0.14 μM and Δ159ChlH K app =0.05±0.03 μM.

    Techniques Used: Activity Assay, Concentration Assay, Binding Assay

    25) Product Images from "A Drosophila Reporter for the Translational Activation of ATF4 Marks Stressed Cells during Development"

    Article Title: A Drosophila Reporter for the Translational Activation of ATF4 Marks Stressed Cells during Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126795

    The expression of ATF4 . 5′UTR > dsRed upon ER stress. 2 nd instar larvae expressing ATF4 reporter were starved for 4 h and then fed with control (A, E, I, M, Q) or 10 μg/mL tunicamycin (B, F, J, N, R) or 1 μM thapsigargin (C, G, K, O, S) or 5 mM DTT (D, H, L, P, T) in Schneider′s Drosophila medium for 5 h. Next, whole mount labeling of tissues were performed with the anti-dsRed antibody. DsRed expressions in salivary gland (E-H), gut (I-L) and fat body (M-P) increased in response to ER stress-inducing agents. In brain (A-D) and mapighian tubule (Q-T), feeding with ER stress-inducing agents did not noticeably alter dsRed expression. The scale bars in (A, E, M) represent 100 μm.
    Figure Legend Snippet: The expression of ATF4 . 5′UTR > dsRed upon ER stress. 2 nd instar larvae expressing ATF4 reporter were starved for 4 h and then fed with control (A, E, I, M, Q) or 10 μg/mL tunicamycin (B, F, J, N, R) or 1 μM thapsigargin (C, G, K, O, S) or 5 mM DTT (D, H, L, P, T) in Schneider′s Drosophila medium for 5 h. Next, whole mount labeling of tissues were performed with the anti-dsRed antibody. DsRed expressions in salivary gland (E-H), gut (I-L) and fat body (M-P) increased in response to ER stress-inducing agents. In brain (A-D) and mapighian tubule (Q-T), feeding with ER stress-inducing agents did not noticeably alter dsRed expression. The scale bars in (A, E, M) represent 100 μm.

    Techniques Used: Expressing, Labeling

    26) Product Images from "Mutations in the MutS? interaction interface of MLH1 can abolish DNA mismatch repair"

    Article Title: Mutations in the MutS? interaction interface of MLH1 can abolish DNA mismatch repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl944

    Crosslinking of E.coli MutL N131C to MutS. ( A ) Crosslinking was performed using E.coli MutS (400 nM), MutL N131C (1000 nM) and a 484 bp DNA substrate (100 nM) in the presence of the indicated nucleotide (1 mM) as detailed in Materials and Methods. BM[PEO] 4 was added to a final concentration of 50 μM and the reaction was incubated for 1 min at 37°C. Crosslinking was quenched by addition of 50 mM DTT, and the products were separated by SDS–PAGE and protein bands were visualized by colloidal Coomassie staining that also stains DNA. Note that the additional band attributed to a MutS–MutL N131C crosslink (S–L N131C ) is only observed in the presence of both ATP and DNA. ( B ) Photocrosslinking of MutL N131C was carried out in the presence or absence of MutH C96S . Both proteins were pre-incubated at 10 and 2.5 μM, respectively, with 500 μM MBP in the presence of 4 mM nucleotide, ATP or AMP–PNP, for 30 min at room temperature. Reactions were stopped by adding DTT. For photocrosslinking, 2 μM modified MutL, 500 nM MutH, 1 μM MutS wt , 0.8 mM nucleotide and 25 nM 484 bp mismatch DNA were irradiated for 25 min at 354 nm. The products were separated by SDS–PAGE and visualized by Coomassie staining without staining the DNA.
    Figure Legend Snippet: Crosslinking of E.coli MutL N131C to MutS. ( A ) Crosslinking was performed using E.coli MutS (400 nM), MutL N131C (1000 nM) and a 484 bp DNA substrate (100 nM) in the presence of the indicated nucleotide (1 mM) as detailed in Materials and Methods. BM[PEO] 4 was added to a final concentration of 50 μM and the reaction was incubated for 1 min at 37°C. Crosslinking was quenched by addition of 50 mM DTT, and the products were separated by SDS–PAGE and protein bands were visualized by colloidal Coomassie staining that also stains DNA. Note that the additional band attributed to a MutS–MutL N131C crosslink (S–L N131C ) is only observed in the presence of both ATP and DNA. ( B ) Photocrosslinking of MutL N131C was carried out in the presence or absence of MutH C96S . Both proteins were pre-incubated at 10 and 2.5 μM, respectively, with 500 μM MBP in the presence of 4 mM nucleotide, ATP or AMP–PNP, for 30 min at room temperature. Reactions were stopped by adding DTT. For photocrosslinking, 2 μM modified MutL, 500 nM MutH, 1 μM MutS wt , 0.8 mM nucleotide and 25 nM 484 bp mismatch DNA were irradiated for 25 min at 354 nm. The products were separated by SDS–PAGE and visualized by Coomassie staining without staining the DNA.

    Techniques Used: Concentration Assay, Incubation, SDS Page, Staining, Modification, Irradiation

    27) Product Images from "TorsinA participates in endoplasmic reticulum-associated degradation"

    Article Title: TorsinA participates in endoplasmic reticulum-associated degradation

    Journal: Nature communications

    doi: 10.1038/ncomms1383

    DYT1 patient fibroblasts are sensitive to ER stress and less able to degrade GFP-CFTRΔF508 (a b) Primary skin fibroblasts from two DYT1 patients (dashed lines; D2551 =Δ; D2306 = ∇) and two controls (solid lines; HF19 = circle; 2131 = square) were infected with a lentivirus vector carrying the expression cassette for Gluc followed 72 h later by exposure to varying concentrations of (a) DTT or (b) 6-OHDA for 4 h. The activity of Gluc in the medium was measured using a luminometer 15 , 34 . DTT treatment results are shown as the mean of 3 experiments ± S.D. at 0.625 mM concentration (where control lines gave essentially equal values). The difference between D2551 and controls was *p
    Figure Legend Snippet: DYT1 patient fibroblasts are sensitive to ER stress and less able to degrade GFP-CFTRΔF508 (a b) Primary skin fibroblasts from two DYT1 patients (dashed lines; D2551 =Δ; D2306 = ∇) and two controls (solid lines; HF19 = circle; 2131 = square) were infected with a lentivirus vector carrying the expression cassette for Gluc followed 72 h later by exposure to varying concentrations of (a) DTT or (b) 6-OHDA for 4 h. The activity of Gluc in the medium was measured using a luminometer 15 , 34 . DTT treatment results are shown as the mean of 3 experiments ± S.D. at 0.625 mM concentration (where control lines gave essentially equal values). The difference between D2551 and controls was *p

    Techniques Used: Infection, Plasmid Preparation, Expressing, Activity Assay, Concentration Assay

    28) Product Images from "Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression"

    Article Title: Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-10-65

    RI protein yields after shake flask EnBase fed-batch cultivation of E. coli ER2566 pET21bRI pGro7 without DTT (-DTT) or with addition of 12 mM of DTT 2 hours after induction of RI (+DTT) . Total (white bars) and soluble (black bars) RI amounts in milligrams per gram of dry cell weight [mg (g CDW) -1 ] (A) and RI activities in normalised crude extracts in kilo units per gram of wet cell weight [kU (g CWW) -1 ] (B) 4 hours after RI induction. The amounts of target protein in (A) were determined from scanned SDS-PAGE gel images as described in material and Methods. Standard deviations represent RI amounts from 3 assays.
    Figure Legend Snippet: RI protein yields after shake flask EnBase fed-batch cultivation of E. coli ER2566 pET21bRI pGro7 without DTT (-DTT) or with addition of 12 mM of DTT 2 hours after induction of RI (+DTT) . Total (white bars) and soluble (black bars) RI amounts in milligrams per gram of dry cell weight [mg (g CDW) -1 ] (A) and RI activities in normalised crude extracts in kilo units per gram of wet cell weight [kU (g CWW) -1 ] (B) 4 hours after RI induction. The amounts of target protein in (A) were determined from scanned SDS-PAGE gel images as described in material and Methods. Standard deviations represent RI amounts from 3 assays.

    Techniques Used: SDS Page

    29) Product Images from "Disulfide-Bond Formation by a Single Cysteine Mutation in Adenovirus Protein VI Impairs Capsid Release and Membrane Lysis"

    Article Title: Disulfide-Bond Formation by a Single Cysteine Mutation in Adenovirus Protein VI Impairs Capsid Release and Membrane Lysis

    Journal: Virology

    doi: 10.1016/j.virol.2012.03.024

    Protein VI molecules are disulfide-linked in VI-G48C particles and purified protein. A) Purified wild type or VI-G48C AdV were boiled in SDS sample buffer ± 2 mM DTT and analyzed by SDS-PAGE and western blot for protein VI. B) Affinity-purified recombinant VI114 proteins were boiled in SDS sample buffer ± 2 mM DTT, loaded on SDS-PAGE gels, and visualized with Simply Blue staining. Dimer (D) and monomer bands (M) are indicated in both panels.
    Figure Legend Snippet: Protein VI molecules are disulfide-linked in VI-G48C particles and purified protein. A) Purified wild type or VI-G48C AdV were boiled in SDS sample buffer ± 2 mM DTT and analyzed by SDS-PAGE and western blot for protein VI. B) Affinity-purified recombinant VI114 proteins were boiled in SDS sample buffer ± 2 mM DTT, loaded on SDS-PAGE gels, and visualized with Simply Blue staining. Dimer (D) and monomer bands (M) are indicated in both panels.

    Techniques Used: Purification, SDS Page, Western Blot, Affinity Purification, Staining

    30) Product Images from "Structural Stability of Human Protein Tyrosine Phosphatase ? Catalytic Domain: Effect of Point Mutations"

    Article Title: Structural Stability of Human Protein Tyrosine Phosphatase ? Catalytic Domain: Effect of Point Mutations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032555

    Intrinsic fluorescence emission spectra of PTPρ wild-type and mutants. Fluorescence spectra of PTPρ wild-type and mutants in 0 M (continuous lines), 8.30 M (dotted lines), 3.95 M (D927G and N1128I, dashed lines) and 4.45 M urea (wild-type and Q987K, dashed lines) were recorded at 0.04 mg/ml protein concentration (295 nm excitation wavelength) at 10°C in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 200 µM DTT.
    Figure Legend Snippet: Intrinsic fluorescence emission spectra of PTPρ wild-type and mutants. Fluorescence spectra of PTPρ wild-type and mutants in 0 M (continuous lines), 8.30 M (dotted lines), 3.95 M (D927G and N1128I, dashed lines) and 4.45 M urea (wild-type and Q987K, dashed lines) were recorded at 0.04 mg/ml protein concentration (295 nm excitation wavelength) at 10°C in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 200 µM DTT.

    Techniques Used: Fluorescence, Protein Concentration

    Effect of urea on near-UV CD spectra of PTPρ wild-type and D927G. ( A ) Near-UV CD spectra of wild-type and D927G in 0 M, 4.28 M and 7.31 M urea were recorded in a 1-cm quartz cuvette at 2.4 mg/ml protein concentration at 10°C in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 2 mM DTT. ( B ) and ( C ) Near-UV CD changes of wild-type and D927G at increasing urea concentrations reported as the first (V1, B ) and the second (V2, C ) column of the V matrix. V1 and V2 were obtained by SVD of the near-UV CD spectral data as described in the text.
    Figure Legend Snippet: Effect of urea on near-UV CD spectra of PTPρ wild-type and D927G. ( A ) Near-UV CD spectra of wild-type and D927G in 0 M, 4.28 M and 7.31 M urea were recorded in a 1-cm quartz cuvette at 2.4 mg/ml protein concentration at 10°C in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 2 mM DTT. ( B ) and ( C ) Near-UV CD changes of wild-type and D927G at increasing urea concentrations reported as the first (V1, B ) and the second (V2, C ) column of the V matrix. V1 and V2 were obtained by SVD of the near-UV CD spectral data as described in the text.

    Techniques Used: Protein Concentration

    Spectroscopic properties of PTPρ wild-type and mutants. ( A ) Near-UV CD spectra were recorded in a 1-cm quartz cuvette at 1.0 mg/ml protein concentration in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 2 mM DTT. ( B ) Intrinsic fluorescence emission spectra were recorded at 0.04 mg/ml protein concentration (295 nm excitation wavelength) in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 200 µM DTT. ( C ) Far-UV CD spectra were recorded in a 0.1-cm quartz cuvette at 0.2 mg/ml in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 0.4 mM DTT.
    Figure Legend Snippet: Spectroscopic properties of PTPρ wild-type and mutants. ( A ) Near-UV CD spectra were recorded in a 1-cm quartz cuvette at 1.0 mg/ml protein concentration in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 2 mM DTT. ( B ) Intrinsic fluorescence emission spectra were recorded at 0.04 mg/ml protein concentration (295 nm excitation wavelength) in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 200 µM DTT. ( C ) Far-UV CD spectra were recorded in a 0.1-cm quartz cuvette at 0.2 mg/ml in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 0.4 mM DTT.

    Techniques Used: Protein Concentration, Fluorescence

    Thermal transition of PTPρ wild-type and mutants. ( A ) PTPρ wild-type, N1128I, Q987K and D927G were heated from 10°C to 72°C in a 0.1-cm quartz cuvette at 0.2 mg/ml in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 0.4 mM DTT. The dichroic activity at 209 nm was monitored continuously every 0.5°C. The inset shows the first derivative of the same data. ( B ) PMTV data recorded in the same experiments shown in ( A ).
    Figure Legend Snippet: Thermal transition of PTPρ wild-type and mutants. ( A ) PTPρ wild-type, N1128I, Q987K and D927G were heated from 10°C to 72°C in a 0.1-cm quartz cuvette at 0.2 mg/ml in 20 mM Tris/HCl, pH 7.5 containing 0.2 M NaCl and 0.4 mM DTT. The dichroic activity at 209 nm was monitored continuously every 0.5°C. The inset shows the first derivative of the same data. ( B ) PMTV data recorded in the same experiments shown in ( A ).

    Techniques Used: Activity Assay

    31) Product Images from "Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies Themselves"

    Article Title: Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies Themselves

    Journal: ACS chemical biology

    doi: 10.1021/acschembio.8b00729

    Temporal change in β -sheet content of WT or G93A-SOD1 measured using ThT fluorescence. 40 μ M of WT (A, C) or G93A- (B, D) SOD1 were incubated at 37 °C with fast agitation in the absence (A, B) or presence (C, D) of 25 mM DTT and the change in ThT fluorescence was monitored in a plate reader for 40 h. To facilitate comparison, all the reactions are shown with the same scale in the y -axis. In panels A and B, the insets show the small changes in fluorescence using adjusted y -axis scales. In panels C and D, insets show one reaction with the lag phase (L), first small increase (I1), first plateau (P1), second large increase (I2), and final plateau (P2) indicated.
    Figure Legend Snippet: Temporal change in β -sheet content of WT or G93A-SOD1 measured using ThT fluorescence. 40 μ M of WT (A, C) or G93A- (B, D) SOD1 were incubated at 37 °C with fast agitation in the absence (A, B) or presence (C, D) of 25 mM DTT and the change in ThT fluorescence was monitored in a plate reader for 40 h. To facilitate comparison, all the reactions are shown with the same scale in the y -axis. In panels A and B, the insets show the small changes in fluorescence using adjusted y -axis scales. In panels C and D, insets show one reaction with the lag phase (L), first small increase (I1), first plateau (P1), second large increase (I2), and final plateau (P2) indicated.

    Techniques Used: Fluorescence, Incubation

    Native-PAGE/Western blot analysis of WT and G93A-SOD1 during aggregation. 40 μ M of WT (A, C, E) or G93A- (B, D, F) SOD1 were incubated at 37 °C with fast agitation in the presence of 25 mM DTT. Aliquots were taken every 4 h, flash frozen, and stored at −80 °C. At the end of the reactions, the aliquots were fractionated by native PAGE, and the proteins were transferred to nitrocellulose membranes and probed with each antibody. Only Ab16831 (A,B), 2770 (C,D), and B8H10 yielded sufficient signal of signal-to-noise ratio and therefore the other antibodies are not shown. NR = nonreduced. Positions of molecular-weight markers are shown on the left of each membrane.
    Figure Legend Snippet: Native-PAGE/Western blot analysis of WT and G93A-SOD1 during aggregation. 40 μ M of WT (A, C, E) or G93A- (B, D, F) SOD1 were incubated at 37 °C with fast agitation in the presence of 25 mM DTT. Aliquots were taken every 4 h, flash frozen, and stored at −80 °C. At the end of the reactions, the aliquots were fractionated by native PAGE, and the proteins were transferred to nitrocellulose membranes and probed with each antibody. Only Ab16831 (A,B), 2770 (C,D), and B8H10 yielded sufficient signal of signal-to-noise ratio and therefore the other antibodies are not shown. NR = nonreduced. Positions of molecular-weight markers are shown on the left of each membrane.

    Techniques Used: Clear Native PAGE, Western Blot, Incubation, Molecular Weight

    ThT fluorescence and antibody reactivity for E100K-SOD1. 40 μ M of E100 K-SOD1 were incubated at 37 °C with fast agitation in the presence of 25 mM DTT. (A) The change in ThT fluorescence was monitored in a plate reader for 40 h. (B) Representative dot blots with each antibody. (C) Averaged values from densitometric analysis of two independent experiments including a total of four replicates. The lines are added to help guide the eye but do not imply that the points should be connected.
    Figure Legend Snippet: ThT fluorescence and antibody reactivity for E100K-SOD1. 40 μ M of E100 K-SOD1 were incubated at 37 °C with fast agitation in the presence of 25 mM DTT. (A) The change in ThT fluorescence was monitored in a plate reader for 40 h. (B) Representative dot blots with each antibody. (C) Averaged values from densitometric analysis of two independent experiments including a total of four replicates. The lines are added to help guide the eye but do not imply that the points should be connected.

    Techniques Used: Fluorescence, Incubation

    32) Product Images from "Therapeutic Anti-Methamphetamine Antibody Fragment-Nanoparticle Conjugates: Synthesis and In Vitro Characterization"

    Article Title: Therapeutic Anti-Methamphetamine Antibody Fragment-Nanoparticle Conjugates: Synthesis and In Vitro Characterization

    Journal: Bioconjugate chemistry

    doi: 10.1021/bc300204n

    A. SEC analysis of dendribodies before purification and DTT treated scFv6H4Cys. The solid and dotted lines represents the dendribodies and scFv6H4Cys respectively. DTT treated scFv6H4Cys exists in vitro mainly as a monomer shown by prominent peak at 27
    Figure Legend Snippet: A. SEC analysis of dendribodies before purification and DTT treated scFv6H4Cys. The solid and dotted lines represents the dendribodies and scFv6H4Cys respectively. DTT treated scFv6H4Cys exists in vitro mainly as a monomer shown by prominent peak at 27

    Techniques Used: Size-exclusion Chromatography, Purification, In Vitro

    33) Product Images from "Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair"

    Article Title: Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku288

    Site-directed alterations and iodoacetamide treatment of PARP-1 reduce intrinsic covalent cross-linking of PARP-1. ( a ) A phosphorimage of cross-linked PARP-1 to 32 P-labeled AP site-containing DNA is shown. PARP-1 (10 μM) was incubated with 50 mM iodoacetamide in a reaction mixture (10 μl) containing 25 mM NaPO 4 buffer, pH 7.0 and 25 mM NaCl for 30 min at room temperature in the dark. Then, the addition of 60 mM DTT was used to terminate the reaction. The excess amounts of iodoacetamide and DTT were removed with a Zebra micro spin desalting column. The intrinsic cross-linking reaction was performed with untreated PARP-1 (lane 1) or iodoacetamide-treated PARP-1 (lane 2). After capturing the phosphorimage of the 32 P-labeled DNA-PARP-1 complex (upper panel), the same gel was stained with Coomassie-blue for proteins (lower panel). ( b ) Photographs of the phosphorimage and the Coomassie-stained gel are shown. The cross-linking reaction was performed with wild-type (lane 1), single mutant C256A (lane 2) or with a double mutant C256A/C845A (lane 3) of PARP-1 as in panel (a). The results revealed a significant reduction in cross-linking of PARP-1 with the iodoactamide-treated sample or with the alteration of solvent exposed cysteine residues 256 and 845 to alanines.
    Figure Legend Snippet: Site-directed alterations and iodoacetamide treatment of PARP-1 reduce intrinsic covalent cross-linking of PARP-1. ( a ) A phosphorimage of cross-linked PARP-1 to 32 P-labeled AP site-containing DNA is shown. PARP-1 (10 μM) was incubated with 50 mM iodoacetamide in a reaction mixture (10 μl) containing 25 mM NaPO 4 buffer, pH 7.0 and 25 mM NaCl for 30 min at room temperature in the dark. Then, the addition of 60 mM DTT was used to terminate the reaction. The excess amounts of iodoacetamide and DTT were removed with a Zebra micro spin desalting column. The intrinsic cross-linking reaction was performed with untreated PARP-1 (lane 1) or iodoacetamide-treated PARP-1 (lane 2). After capturing the phosphorimage of the 32 P-labeled DNA-PARP-1 complex (upper panel), the same gel was stained with Coomassie-blue for proteins (lower panel). ( b ) Photographs of the phosphorimage and the Coomassie-stained gel are shown. The cross-linking reaction was performed with wild-type (lane 1), single mutant C256A (lane 2) or with a double mutant C256A/C845A (lane 3) of PARP-1 as in panel (a). The results revealed a significant reduction in cross-linking of PARP-1 with the iodoactamide-treated sample or with the alteration of solvent exposed cysteine residues 256 and 845 to alanines.

    Techniques Used: Labeling, Incubation, Staining, Mutagenesis

    34) Product Images from "Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair"

    Article Title: Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku288

    Site-directed alterations and iodoacetamide treatment of PARP-1 reduce intrinsic covalent cross-linking of PARP-1. ( a ) A phosphorimage of cross-linked PARP-1 to 32 P-labeled AP site-containing DNA is shown. PARP-1 (10 μM) was incubated with 50 mM iodoacetamide in a reaction mixture (10 μl) containing 25 mM NaPO 4 buffer, pH 7.0 and 25 mM NaCl for 30 min at room temperature in the dark. Then, the addition of 60 mM DTT was used to terminate the reaction. The excess amounts of iodoacetamide and DTT were removed with a Zebra micro spin desalting column. The intrinsic cross-linking reaction was performed with untreated PARP-1 (lane 1) or iodoacetamide-treated PARP-1 (lane 2). After capturing the phosphorimage of the 32 P-labeled DNA-PARP-1 complex (upper panel), the same gel was stained with Coomassie-blue for proteins (lower panel). ( b ) Photographs of the phosphorimage and the Coomassie-stained gel are shown. The cross-linking reaction was performed with wild-type (lane 1), single mutant C256A (lane 2) or with a double mutant C256A/C845A (lane 3) of PARP-1 as in panel (a). The results revealed a significant reduction in cross-linking of PARP-1 with the iodoactamide-treated sample or with the alteration of solvent exposed cysteine residues 256 and 845 to alanines.
    Figure Legend Snippet: Site-directed alterations and iodoacetamide treatment of PARP-1 reduce intrinsic covalent cross-linking of PARP-1. ( a ) A phosphorimage of cross-linked PARP-1 to 32 P-labeled AP site-containing DNA is shown. PARP-1 (10 μM) was incubated with 50 mM iodoacetamide in a reaction mixture (10 μl) containing 25 mM NaPO 4 buffer, pH 7.0 and 25 mM NaCl for 30 min at room temperature in the dark. Then, the addition of 60 mM DTT was used to terminate the reaction. The excess amounts of iodoacetamide and DTT were removed with a Zebra micro spin desalting column. The intrinsic cross-linking reaction was performed with untreated PARP-1 (lane 1) or iodoacetamide-treated PARP-1 (lane 2). After capturing the phosphorimage of the 32 P-labeled DNA-PARP-1 complex (upper panel), the same gel was stained with Coomassie-blue for proteins (lower panel). ( b ) Photographs of the phosphorimage and the Coomassie-stained gel are shown. The cross-linking reaction was performed with wild-type (lane 1), single mutant C256A (lane 2) or with a double mutant C256A/C845A (lane 3) of PARP-1 as in panel (a). The results revealed a significant reduction in cross-linking of PARP-1 with the iodoactamide-treated sample or with the alteration of solvent exposed cysteine residues 256 and 845 to alanines.

    Techniques Used: Labeling, Incubation, Staining, Mutagenesis

    35) Product Images from "A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases"

    Article Title: A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2015.00068

    PTEN CiV is inhibited by bisperoxovanadate compounds. (A) Bars representing the effect of overnight incubation of bisperoxovanadate compounds on PTEN CiV activity. Fluorescence intensity was acquired with TIRF microscopy of HEK cells expressing PTEN CiV , TRPV1 channel, and Akt-PH-GFP. Decrease in the fluorescence signal recorded from the membrane is observed upon capsaicin application. Cells were treated with different concentrations of the PTEN inhibitors bpV(phen) or bpV(Hopic). Cells that after an overnight incubation with 10 μM bpV(phen) were washed out and incubated for 3 h with 5 mM of the reducing agent DTT showed a partial recovery of phosphatase activity. (B–D) Graph demonstrating the effect of the inhibitors in real time. Data is normalized to the time intervals immediately preceding each capsaicin application. See Supplementary Figure 5 for a presentation of the data without re-normalization for the second agonist application. (B) HEK cells that had initially responded to a 100 nM capsaicin activation were incubated for 30 min with 200 μM of the inhibitor bpV(phen). After the 30 min incubation they presented no response to a second capsaicin application ( n = 11, e = 4). (C) Application of the inhibitor bpV(Hopic) also impairs PTEN CiV activity ( n = 10, e = 4). (D) Graph showing a control experiment where no inhibitor was applied between the two capsaicin applications ( n = 9, e = 3). Statistically significant difference between control and treated samples is marked with * , while the # sign indicates significant difference between the 10 μM bpV(phen) and the DTT treated samples. Numbers in columns represent the number of cells.
    Figure Legend Snippet: PTEN CiV is inhibited by bisperoxovanadate compounds. (A) Bars representing the effect of overnight incubation of bisperoxovanadate compounds on PTEN CiV activity. Fluorescence intensity was acquired with TIRF microscopy of HEK cells expressing PTEN CiV , TRPV1 channel, and Akt-PH-GFP. Decrease in the fluorescence signal recorded from the membrane is observed upon capsaicin application. Cells were treated with different concentrations of the PTEN inhibitors bpV(phen) or bpV(Hopic). Cells that after an overnight incubation with 10 μM bpV(phen) were washed out and incubated for 3 h with 5 mM of the reducing agent DTT showed a partial recovery of phosphatase activity. (B–D) Graph demonstrating the effect of the inhibitors in real time. Data is normalized to the time intervals immediately preceding each capsaicin application. See Supplementary Figure 5 for a presentation of the data without re-normalization for the second agonist application. (B) HEK cells that had initially responded to a 100 nM capsaicin activation were incubated for 30 min with 200 μM of the inhibitor bpV(phen). After the 30 min incubation they presented no response to a second capsaicin application ( n = 11, e = 4). (C) Application of the inhibitor bpV(Hopic) also impairs PTEN CiV activity ( n = 10, e = 4). (D) Graph showing a control experiment where no inhibitor was applied between the two capsaicin applications ( n = 9, e = 3). Statistically significant difference between control and treated samples is marked with * , while the # sign indicates significant difference between the 10 μM bpV(phen) and the DTT treated samples. Numbers in columns represent the number of cells.

    Techniques Used: Incubation, Activity Assay, Fluorescence, Microscopy, Expressing, Activation Assay

    36) Product Images from "What interactions drive the salivary mucosal pellicle formation?"

    Article Title: What interactions drive the salivary mucosal pellicle formation?

    Journal: Colloids and Surfaces. B, Biointerfaces

    doi: 10.1016/j.colsurfb.2014.05.020

    Gels following CBB and PAS staining (WMS only) of WMS and PS pellicle formations on hydrophobic particles from different subjects. Lanes: saliva pre (1), saliva post (2), water wash 1 (3), water wash 2 (4), SDS 1 (5), SDS 2 (6) and boiled DTT, LDS and water (7). Boxes highlight clear differences in binding ± TGM. The box highlighted by the arrow indicates an effect only seen in 50% of the samples. It shows what appear to be a possible cross-linked proteins, perhaps statherin and histatin bound higher due to crosslinking.
    Figure Legend Snippet: Gels following CBB and PAS staining (WMS only) of WMS and PS pellicle formations on hydrophobic particles from different subjects. Lanes: saliva pre (1), saliva post (2), water wash 1 (3), water wash 2 (4), SDS 1 (5), SDS 2 (6) and boiled DTT, LDS and water (7). Boxes highlight clear differences in binding ± TGM. The box highlighted by the arrow indicates an effect only seen in 50% of the samples. It shows what appear to be a possible cross-linked proteins, perhaps statherin and histatin bound higher due to crosslinking.

    Techniques Used: Staining, Binding Assay

    37) Product Images from "Quantitative Proteomics Employing Primary Amine Affinity Tags"

    Article Title: Quantitative Proteomics Employing Primary Amine Affinity Tags

    Journal: Journal of Biomolecular Techniques : JBT

    doi:

    MALDI-TOF/TOF spectra of bradykinin control. Arrows denote bradykinin peaks. Bradykinin (A) was labeled with Sulfo-NHS-SS-biotin (B) . After application of the sample to a streptavidin column, the peptide was retrieved with the addition of 5 mM DTT. The biotin moiety and part of the linker arm were retained on the column (C) . The resulting free thiol on the labeled peptide was methylated with methyl iodide (D) .
    Figure Legend Snippet: MALDI-TOF/TOF spectra of bradykinin control. Arrows denote bradykinin peaks. Bradykinin (A) was labeled with Sulfo-NHS-SS-biotin (B) . After application of the sample to a streptavidin column, the peptide was retrieved with the addition of 5 mM DTT. The biotin moiety and part of the linker arm were retained on the column (C) . The resulting free thiol on the labeled peptide was methylated with methyl iodide (D) .

    Techniques Used: Labeling, Methylation

    38) Product Images from "Aptamer-functionalized peptide H3CR5C as a novel nanovehicle for codelivery of fasudil and miRNA-195 targeting hepatocellular carcinoma"

    Article Title: Aptamer-functionalized peptide H3CR5C as a novel nanovehicle for codelivery of fasudil and miRNA-195 targeting hepatocellular carcinoma

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S108128

    Release profiles of plain fasudil and DTT-triggered fasudil from Fasudil SHP miR195 . Note: Data are shown as the mean ± SD (n=3). Abbreviations: DTT, dithiothreitol; SD, standard deviation; h, hour; miR195, miRNA-195.
    Figure Legend Snippet: Release profiles of plain fasudil and DTT-triggered fasudil from Fasudil SHP miR195 . Note: Data are shown as the mean ± SD (n=3). Abbreviations: DTT, dithiothreitol; SD, standard deviation; h, hour; miR195, miRNA-195.

    Techniques Used: Standard Deviation

    Agarose gel electrophoresis. Notes: ( A ) Condensation ability of miR195 with Fasudil SHP miR195 and DTT-triggered miR195 release from Fasudil SHP miR195 . ( B ) Stability test of Fasudil SHP miR195 in 50% FBS conditions at 37°C. Abbreviations: DTT, dithiothreitol; FBS, fetal bovine serum; h, hour; miR195, miRNA-195.
    Figure Legend Snippet: Agarose gel electrophoresis. Notes: ( A ) Condensation ability of miR195 with Fasudil SHP miR195 and DTT-triggered miR195 release from Fasudil SHP miR195 . ( B ) Stability test of Fasudil SHP miR195 in 50% FBS conditions at 37°C. Abbreviations: DTT, dithiothreitol; FBS, fetal bovine serum; h, hour; miR195, miRNA-195.

    Techniques Used: Agarose Gel Electrophoresis

    39) Product Images from "Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins"

    Article Title: Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201404083

    Disulfide bonds in MagT1-dependent substrates. (A) HeLa cells treated with NC or MagT1 siRNA were treated with 3 mM DTT for 5 min before a 5-min pulse, 10-min chase labeling period. Endogenous cathepsin C was immunoprecipitated using anti-CatC sera and resolved by SDS-PAGE. Diagrams of pCatCΔ234-HA (B) and FVII N183Q (D) showing the signal sequence (black), glycosylation sites, disulfide bonds (red lines), free cysteine residues (diamonds), mature protein domains (green, cyan, magenta, and yellow segments), and the C-terminal HA tag on pCatCΔ234-HA. Disulfides that link (pCatCΔ234) or bracket (FVII N183Q) a STT3B-dependent glycosylation site are indicated. (C and E) HeLa cells were treated with NC, or siRNAs specific for STT3A, STT3B, or MagT1 for 48 h as indicated, then transfected with pCatCΔ234-HA (C) or FVII N183Q (E and F) expression vectors and cultured for an additional 24 h before pulse labeling. Cells were pulse labeled for 4 min (C), pulse labeled for 2 min, and chased for 30 min (E), or pulsed for 2 min and chased as indicated (F). Glycoprotein substrates were precipitated with anti-HA sera (C) or anti-factor VII sera (E and F). Quantified values below gel lanes (A, C, and E) are for the displayed image that is representative of two or more experiments. Data points in F are the mean of two determinations, with individual data points indicated by error bars.
    Figure Legend Snippet: Disulfide bonds in MagT1-dependent substrates. (A) HeLa cells treated with NC or MagT1 siRNA were treated with 3 mM DTT for 5 min before a 5-min pulse, 10-min chase labeling period. Endogenous cathepsin C was immunoprecipitated using anti-CatC sera and resolved by SDS-PAGE. Diagrams of pCatCΔ234-HA (B) and FVII N183Q (D) showing the signal sequence (black), glycosylation sites, disulfide bonds (red lines), free cysteine residues (diamonds), mature protein domains (green, cyan, magenta, and yellow segments), and the C-terminal HA tag on pCatCΔ234-HA. Disulfides that link (pCatCΔ234) or bracket (FVII N183Q) a STT3B-dependent glycosylation site are indicated. (C and E) HeLa cells were treated with NC, or siRNAs specific for STT3A, STT3B, or MagT1 for 48 h as indicated, then transfected with pCatCΔ234-HA (C) or FVII N183Q (E and F) expression vectors and cultured for an additional 24 h before pulse labeling. Cells were pulse labeled for 4 min (C), pulse labeled for 2 min, and chased for 30 min (E), or pulsed for 2 min and chased as indicated (F). Glycoprotein substrates were precipitated with anti-HA sera (C) or anti-factor VII sera (E and F). Quantified values below gel lanes (A, C, and E) are for the displayed image that is representative of two or more experiments. Data points in F are the mean of two determinations, with individual data points indicated by error bars.

    Techniques Used: Labeling, Immunoprecipitation, SDS Page, Sequencing, Transfection, Expressing, Cell Culture

    Formation of mixed disulfides between MagT1 and glycoprotein substrates. (A) Diagram of the pCatC-Insert-Δ234 construct. (B–D) HeLa cells were treated with the NC or MagT1 siRNA for 48 h before cotransfection with wild-type or mutant versions of the pCatCΔ234-HA (B and C), FVII N183Q (D), and MagT1-V5 expression vectors (B–D). The m1, m2, and m3 mutants of MagT1 are defined in Fig. 5 A . Cells were pulse labeled for 4 min (A–D) and chased for 10 min (B and C) or 40 min (D). Glycoproteins were immunoprecipitated with anti-HA sera (B and C) or anti-FVII sera (D) and quantified after SDS-PAGE. (E) Cells expressing wild-type or mutant versions of MagT1-V5 were treated with NEM to prevent disulfide exchange during cell lysis and sample preparation. Total cell extracts were resolved by nonreducing (−DTT) or reducing (+DTT) SDS-PAGE as indicated, and analyzed by protein immunoblotting using anti-V5 sera. (F) In vivo redox status of MagT1 and PDI in HeLa cells was assayed using a maleimide-shift protocol. The arrows designate oxidized and reduced forms of MagT1 and PDI. A minor MagT1 reactive band in the DPS-oxidized lane (asterisk) is probably due to inefficient formation of a disulfide between cysteine residues located on the cytoplasmic face of TM3 and TM4 (see Fig. 5 A for a map of MagT1 cysteine residues). Quantified values below gel lanes (B–D) are for the displayed image, which is representative of two experiments.
    Figure Legend Snippet: Formation of mixed disulfides between MagT1 and glycoprotein substrates. (A) Diagram of the pCatC-Insert-Δ234 construct. (B–D) HeLa cells were treated with the NC or MagT1 siRNA for 48 h before cotransfection with wild-type or mutant versions of the pCatCΔ234-HA (B and C), FVII N183Q (D), and MagT1-V5 expression vectors (B–D). The m1, m2, and m3 mutants of MagT1 are defined in Fig. 5 A . Cells were pulse labeled for 4 min (A–D) and chased for 10 min (B and C) or 40 min (D). Glycoproteins were immunoprecipitated with anti-HA sera (B and C) or anti-FVII sera (D) and quantified after SDS-PAGE. (E) Cells expressing wild-type or mutant versions of MagT1-V5 were treated with NEM to prevent disulfide exchange during cell lysis and sample preparation. Total cell extracts were resolved by nonreducing (−DTT) or reducing (+DTT) SDS-PAGE as indicated, and analyzed by protein immunoblotting using anti-V5 sera. (F) In vivo redox status of MagT1 and PDI in HeLa cells was assayed using a maleimide-shift protocol. The arrows designate oxidized and reduced forms of MagT1 and PDI. A minor MagT1 reactive band in the DPS-oxidized lane (asterisk) is probably due to inefficient formation of a disulfide between cysteine residues located on the cytoplasmic face of TM3 and TM4 (see Fig. 5 A for a map of MagT1 cysteine residues). Quantified values below gel lanes (B–D) are for the displayed image, which is representative of two experiments.

    Techniques Used: Construct, Cotransfection, Mutagenesis, Expressing, Labeling, Immunoprecipitation, SDS Page, Lysis, Sample Prep, In Vivo

    40) Product Images from "Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe\u2013S center"

    Article Title: Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe\u2013S center

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.06.021

    OA-NO 2 induces iron release from Rbx domain. Non-protein-bound iron present in control and OA-NO 2 -treated PknG samples was recovered, reduced with DTT, and quantified as Fe 2+ using bathophenantrolinedisulfonic acid. The complex absorbs at 535 nm ( ε
    Figure Legend Snippet: OA-NO 2 induces iron release from Rbx domain. Non-protein-bound iron present in control and OA-NO 2 -treated PknG samples was recovered, reduced with DTT, and quantified as Fe 2+ using bathophenantrolinedisulfonic acid. The complex absorbs at 535 nm ( ε

    Techniques Used:

    41) Product Images from "Sulfhydration mediates neuroprotective actions of parkin"

    Article Title: Sulfhydration mediates neuroprotective actions of parkin

    Journal: Nature communications

    doi: 10.1038/ncomms2623

    Parkin is physiologically sulfhydrated ( a ) Parkin expressed in HEK293 cells is sulfhydrated by the H 2 S donor NaHS as detected by the modified biotin switch method. ( b ) Endogenous parkin is basally sulfhydrated in both mouse brain and rat striatum as detected by the maleimide assay in which loss of red fluorescence signal following DTT treatment indicates sulfhydration of the protein. ( c ) Sulfhydration of myc-parkin overexpressed in HEK293 cells is enhanced almost 20 fold upon overexpression of GST-CBS, one of the principle H 2 S producers in the brain. n=3, P
    Figure Legend Snippet: Parkin is physiologically sulfhydrated ( a ) Parkin expressed in HEK293 cells is sulfhydrated by the H 2 S donor NaHS as detected by the modified biotin switch method. ( b ) Endogenous parkin is basally sulfhydrated in both mouse brain and rat striatum as detected by the maleimide assay in which loss of red fluorescence signal following DTT treatment indicates sulfhydration of the protein. ( c ) Sulfhydration of myc-parkin overexpressed in HEK293 cells is enhanced almost 20 fold upon overexpression of GST-CBS, one of the principle H 2 S producers in the brain. n=3, P

    Techniques Used: Modification, Fluorescence, Over Expression

    42) Product Images from "Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli"

    Article Title: Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

    Journal: Current protocols in protein science / editorial board, John E. Coligan ... [et al.]

    doi: 10.1002/0471140864.ps0603s70

    Gel filtration of an extract containing HIV-1 protease, using Superdex 200 in 4 M guanidine·HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris·Cl (pH 7.5)/4 mM guanidine·Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr). The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) was further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS-PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).
    Figure Legend Snippet: Gel filtration of an extract containing HIV-1 protease, using Superdex 200 in 4 M guanidine·HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris·Cl (pH 7.5)/4 mM guanidine·Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr). The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) was further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS-PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).

    Techniques Used: Filtration, Flow Cytometry, Purification, SDS Page, Migration

    43) Product Images from "Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss"

    Article Title: Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-014-0111-y

    RP-HPLC profiles for the H3 rHA proteins. Each H3 rHA was analyzed in duplicate. Representative chromatograms from the day 0 and 28 time points are provided. rHAs were incubated with 25 mM DTT for at least 30 minutes prior to analysis. ~25 μg of each rHA was injected onto a Poros R1 column and an acetonitrile gradient applied.
    Figure Legend Snippet: RP-HPLC profiles for the H3 rHA proteins. Each H3 rHA was analyzed in duplicate. Representative chromatograms from the day 0 and 28 time points are provided. rHAs were incubated with 25 mM DTT for at least 30 minutes prior to analysis. ~25 μg of each rHA was injected onto a Poros R1 column and an acetonitrile gradient applied.

    Techniques Used: High Performance Liquid Chromatography, Incubation, Injection

    44) Product Images from "Synthetic substrates for measuring activity of autophagy proteases"

    Article Title: Synthetic substrates for measuring activity of autophagy proteases

    Journal: Autophagy

    doi: 10.4161/auto.6.7.13075

    Characterization of Atg4B activity using synthetic peptide substrates. (A) Various concentrations of recombinant Atg4B were incubated with 100 µM synthetic tetrapeptides conjugated with AFC in 50 mM Tris-HCl, pH 8.0, 5 mM DTT at 37°C for
    Figure Legend Snippet: Characterization of Atg4B activity using synthetic peptide substrates. (A) Various concentrations of recombinant Atg4B were incubated with 100 µM synthetic tetrapeptides conjugated with AFC in 50 mM Tris-HCl, pH 8.0, 5 mM DTT at 37°C for

    Techniques Used: Activity Assay, Recombinant, Incubation

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    Article Snippet: .. After 5 min of incubation at 65°C, samples were chilled on ice at least during 1 min, and a reverse transcription (RT) mix containing 44 μl of 5× first-strand buffer (Invitrogen), 22 μl of dithiothreitol (DTT) (0.1 M; Invitrogen), 2 μl of SuperScript III Retrotranscriptase (200 U/μl; Invitrogen), and 1 μl of RNase Out (40 U/μl; Invitrogen) was added to each preparation. cDNA was obtained after a cycle of 10 min at 25°C, 50 min at 50°C, and 5 min at 85°C. .. After the tubes were chilled on ice, untranscribed RNA was eliminated by the addition of 1 μl of RNase H (10 U/μl; Invitrogen) and incubation for 20 min at 37°C.

    Article Title: 5-aza-2′-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells
    Article Snippet: .. Each reaction consisted of 1 μg of total RNA, 4 μl of 5x RT buffer, 2 μl of 0.1 M dithiothreitol (DTT), 0.5 μl of 0.5 μg/μl oligo(dT) primers (Invitrogen), 0.5 μl of 0.5 μg/μl single-stranded random hexanucleotides (Bioron), 1 μl of 10 mM dNTPs (Invitrogen) and 0.5 μl of SuperScript® II Reverse Transcriptase (200 U/μl), and was incubated at 37°C for 15 minutes, at 42°C for 60 minutes and at 90°C for 5 minutes. .. Quantitative PCR (qPCR) was performed utilizing the Applied Biosystems StepOne™ Real-Time PCR system using Absolute qPCR SYBR Green ROX Mix (Thermo Scientific).

    Article Title: Magnetospirillum bellicus sp. nov., a Novel Dissimilatory Perchlorate-Reducing Alphaproteobacterium Isolated from a Bioelectrical Reactor ▿
    Article Snippet: .. To this, 4 μl of 5× First-Strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (DTT) (Invitrogen), and 1 μl RNAseOUT (Invitrogen) were added, mixed gently, and incubated at 42°C for 2 min. Superscript II Reverse Transcriptase (0.5 μl; Invitrogen) was added, mixed gently, and incubated at 42°C for 50 min and then again at 70°C for 15 min. ..

    Article Title: SNIP1 Recruits TET2 to Regulate c-MYC Target Genes and Cellular DNA Damage Response
    Article Snippet: .. 10 mM Dithiothreitol (DTT) was added and incubated for 30 minutes at 60°C, followed by addition of methyl methanethiosulfonate (MMTS) (ThermoFisher Scientific, Waltham, MA) to 20 mM. .. After 30 min incubation in the dark at room temperature, excess MMTS was quenched by addition of 20 mM DTT.

    Article Title: Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile
    Article Snippet: cDNA was synthesized from 2.5 μg of total RNA by adding 1 μg N6 random primer, 10 mM deoxynucleoside triphosphates, 5× buffer, 100 mM dithiothreitol (DTT), 40 U RNase inhibitor (Life Technologies, Burlington, ON, Canada) in a total volume of 50 μl. .. Following incubation at 42°C for 2 min, 1 μl Superscript II (Life Technologies) was added and the reaction mixture was incubated for 1 h at 42°C.

    Amplification:

    Article Title: 5-aza-2′-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells
    Article Snippet: Each reaction consisted of 1 μg of total RNA, 4 μl of 5x RT buffer, 2 μl of 0.1 M dithiothreitol (DTT), 0.5 μl of 0.5 μg/μl oligo(dT) primers (Invitrogen), 0.5 μl of 0.5 μg/μl single-stranded random hexanucleotides (Bioron), 1 μl of 10 mM dNTPs (Invitrogen) and 0.5 μl of SuperScript® II Reverse Transcriptase (200 U/μl), and was incubated at 37°C for 15 minutes, at 42°C for 60 minutes and at 90°C for 5 minutes. .. Primers used for PCR amplification are listed in Table .

    Activity Assay:

    Article Title: Licochalcone A, a Polyphenol Present in Licorice, Suppresses UV-Induced COX-2 Expression by Targeting PI3K, MEK1, and B-Raf
    Article Snippet: .. When the cells reached 80%–90% confluence, they were starved by culturing in 0.1% FBS MEM for an additional 24 h. The cells were then treated for 1 h with LicoA (0–10 μM) or Gc (0–10 μM) and irradiated with sUV for 6 h. After treatment, the cells were disrupted with 100 μL of lysis buffer (0.1 M potassium phosphate buffer (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol (DTT), and 2 mM EDTA), and the level of luciferase activity was measured using a luminometer (Luminoskan Ascent; Thermo Electron, Helsinki, Finland). .. PI3K Assay Active PI3K protein (100 ng) was incubated with LicoA or LY294002 at the indicated concentrations for 10 min at 30 °C.

    Expressing:

    Article Title: 5-aza-2′-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells
    Article Snippet: Paragraph title: E6*I and E7 expression analysis, methylation-specific qPCR and miR-375 detection ... Each reaction consisted of 1 μg of total RNA, 4 μl of 5x RT buffer, 2 μl of 0.1 M dithiothreitol (DTT), 0.5 μl of 0.5 μg/μl oligo(dT) primers (Invitrogen), 0.5 μl of 0.5 μg/μl single-stranded random hexanucleotides (Bioron), 1 μl of 10 mM dNTPs (Invitrogen) and 0.5 μl of SuperScript® II Reverse Transcriptase (200 U/μl), and was incubated at 37°C for 15 minutes, at 42°C for 60 minutes and at 90°C for 5 minutes.

    BIA-KA:

    Article Title: 18O Labeling over a Coffee Break: A Rapid Strategy for Quantitative Proteomics
    Article Snippet: .. Dithiothreitol (DTT) and micro BCA assay kits were purchased from Pierce, Rockford, IL. .. The trypsin spin columns were purchased from Sigma-Aldrich, St. Louis, MO.

    Modification:

    Article Title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study
    Article Snippet: Formic acid (94318) was purchased from Fluka; Trizma® hydrochloride (15506-017), iodoacetiamide (D-6003), dithiothreitol-DL (D-1532), Tween 20 (00–3005), trifluoroethanol (AM9856), sodium chloride (AM9760G) and EDTA (AM9261) were obtained from Ambion. .. Baker, 3003-01); sodium hydroxide (Fisher Scientific, S719932); RapiGest SF surfactant (Waters, 186001861); sequencing grade modified trypsin (Promega, V5111); Pure Proteome Protein A magnetic beads (Millipore, LSKMAGA10) and SA coated paramagnetic beads (Invitrogen, 60210).

    High Performance Liquid Chromatography:

    Article Title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study
    Article Snippet: Acetonitrile (EM-AX0145-1), methanol (BJ230–4), hydrochloric acid (EM-HX0608-7), isopropyl alcohol (BJ323–4), and HPLC grade water (JT9831–3) were obtained from VWR Scientific. .. Formic acid (94318) was purchased from Fluka; Trizma® hydrochloride (15506-017), iodoacetiamide (D-6003), dithiothreitol-DL (D-1532), Tween 20 (00–3005), trifluoroethanol (AM9856), sodium chloride (AM9760G) and EDTA (AM9261) were obtained from Ambion.

    Article Title: 18O Labeling over a Coffee Break: A Rapid Strategy for Quantitative Proteomics
    Article Snippet: Dithiothreitol (DTT) and micro BCA assay kits were purchased from Pierce, Rockford, IL. .. Methanol, acetonitrile, and water were HPLC grade solvents from Burdick & Jackson, Muskegon, MI.

    Article Title: Identification and analysis of phosphorylation status of proteins in dormant terminal buds of poplar
    Article Snippet: Iodoacetamide (IAA) and dithiothreitol (DTT) were purchased from Acros Organics (Morris Plains, NJ, USA). .. HPLC-grade acetonitrile (ACN) was obtained from JT Baker (Thomas Scientific, Swedesboro, NJ, USA).

    Produced:

    Article Title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study
    Article Snippet: Recombinant human PCSK9 (rhuPCSK9) and 1.2A4, a mouse mAb raised specifically to the CDR of RG7652 were also produced at Genentech. .. Formic acid (94318) was purchased from Fluka; Trizma® hydrochloride (15506-017), iodoacetiamide (D-6003), dithiothreitol-DL (D-1532), Tween 20 (00–3005), trifluoroethanol (AM9856), sodium chloride (AM9760G) and EDTA (AM9261) were obtained from Ambion.

    Flow Cytometry:

    Article Title: In-depth Analysis of the Magnaporthe oryzae Conidial Proteome
    Article Snippet: Samples were dried down to 27 μl and 3 μl of 50 mM Dithiothreitol (DTT) (Thermo Fischer Scientific, Rockford, IL) was added to a final DTT concentration of 5 mM. .. This step was repeated one more time and the flow through solvent was discarded before 100 μl of 0.05 M iodoacetamide was added.

    Protease Inhibitor:

    Article Title: Separate mechanisms act concurrently to shed and release the prion protein from the cell
    Article Snippet: Dithiothreitol (DTT) came from USB Corporation. .. Complete protease inhibitor cocktail was from Roche.

    Magnetic Beads:

    Article Title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study
    Article Snippet: Formic acid (94318) was purchased from Fluka; Trizma® hydrochloride (15506-017), iodoacetiamide (D-6003), dithiothreitol-DL (D-1532), Tween 20 (00–3005), trifluoroethanol (AM9856), sodium chloride (AM9760G) and EDTA (AM9261) were obtained from Ambion. .. Baker, 3003-01); sodium hydroxide (Fisher Scientific, S719932); RapiGest SF surfactant (Waters, 186001861); sequencing grade modified trypsin (Promega, V5111); Pure Proteome Protein A magnetic beads (Millipore, LSKMAGA10) and SA coated paramagnetic beads (Invitrogen, 60210).

    Cell Culture:

    Article Title: Tumor Hypoxia Blocks Wnt Processing and Secretion through the Induction of Endoplasmic Reticulum Stress ▿
    Article Snippet: Paragraph title: Cells, cell culture, and reagents. ... Dithiothreitol (DTT) was purchased from Invitrogen.

    Generated:

    Article Title: Tumor Hypoxia Blocks Wnt Processing and Secretion through the Induction of Endoplasmic Reticulum Stress ▿
    Article Snippet: Severe hypoxia was generated in an anaerobic workstation gassed with 5% CO2 , 5% H2 , and 95% N2 containing a palladium catalyst (Sheldon Co., Cornelius, OR). .. Dithiothreitol (DTT) was purchased from Invitrogen.

    Article Title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study
    Article Snippet: RG7652 is a human IgG1 mAb therapeutic generated at Genentech. .. Formic acid (94318) was purchased from Fluka; Trizma® hydrochloride (15506-017), iodoacetiamide (D-6003), dithiothreitol-DL (D-1532), Tween 20 (00–3005), trifluoroethanol (AM9856), sodium chloride (AM9760G) and EDTA (AM9261) were obtained from Ambion.

    Sequencing:

    Article Title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study
    Article Snippet: Formic acid (94318) was purchased from Fluka; Trizma® hydrochloride (15506-017), iodoacetiamide (D-6003), dithiothreitol-DL (D-1532), Tween 20 (00–3005), trifluoroethanol (AM9856), sodium chloride (AM9760G) and EDTA (AM9261) were obtained from Ambion. .. Baker, 3003-01); sodium hydroxide (Fisher Scientific, S719932); RapiGest SF surfactant (Waters, 186001861); sequencing grade modified trypsin (Promega, V5111); Pure Proteome Protein A magnetic beads (Millipore, LSKMAGA10) and SA coated paramagnetic beads (Invitrogen, 60210).

    Article Title: Genetic diversity and drug resistance of HIV-1 among infected pregnant women newly diagnosed in Luanda, Angola
    Article Snippet: Paragraph title: RNA extraction, cDNA synthesis, PCR and sequencing ... The mix contained 25mM DNTP mix, 5X M-MLV buffer, 10mM of dithiothreitol (DTT), 40U of RNase OUT™ (Life Technologies, USA), 0.1mM of MMRTR6 primer ( 5’-TTTTACATCATTAGTGTGGG-3’ ), and 200U of M-MLV enzyme (Life Technologies, USA) [ ].

    Recombinant:

    Article Title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study
    Article Snippet: Recombinant human PCSK9 (rhuPCSK9) and 1.2A4, a mouse mAb raised specifically to the CDR of RG7652 were also produced at Genentech. .. Formic acid (94318) was purchased from Fluka; Trizma® hydrochloride (15506-017), iodoacetiamide (D-6003), dithiothreitol-DL (D-1532), Tween 20 (00–3005), trifluoroethanol (AM9856), sodium chloride (AM9760G) and EDTA (AM9261) were obtained from Ambion.

    Methylation:

    Article Title: 5-aza-2′-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells
    Article Snippet: Paragraph title: E6*I and E7 expression analysis, methylation-specific qPCR and miR-375 detection ... Each reaction consisted of 1 μg of total RNA, 4 μl of 5x RT buffer, 2 μl of 0.1 M dithiothreitol (DTT), 0.5 μl of 0.5 μg/μl oligo(dT) primers (Invitrogen), 0.5 μl of 0.5 μg/μl single-stranded random hexanucleotides (Bioron), 1 μl of 10 mM dNTPs (Invitrogen) and 0.5 μl of SuperScript® II Reverse Transcriptase (200 U/μl), and was incubated at 37°C for 15 minutes, at 42°C for 60 minutes and at 90°C for 5 minutes.

    Isolation:

    Article Title: Magnetospirillum bellicus sp. nov., a Novel Dissimilatory Perchlorate-Reducing Alphaproteobacterium Isolated from a Bioelectrical Reactor ▿
    Article Snippet: Total RNA (7 μl) from the isolation protocol was added to 1 μl of 3 μM cbbM -specific primers ( , ) (Table ), and 4 μl 2.5 mM each deoxynucleoside triphosphate (dNTP) (Invitrogen); heated at 65°C for 5 min; and quickly chilled on ice. .. To this, 4 μl of 5× First-Strand buffer (Invitrogen), 2 μl 0.1 M dithiothreitol (DTT) (Invitrogen), and 1 μl RNAseOUT (Invitrogen) were added, mixed gently, and incubated at 42°C for 2 min. Superscript II Reverse Transcriptase (0.5 μl; Invitrogen) was added, mixed gently, and incubated at 42°C for 50 min and then again at 70°C for 15 min.

    Transfection:

    Article Title: Licochalcone A, a Polyphenol Present in Licorice, Suppresses UV-Induced COX-2 Expression by Targeting PI3K, MEK1, and B-Raf
    Article Snippet: Luciferase Assay for activator protein 1 AP-1 Transactivation Confluent monolayers of HaCaT cells that were stably transfected with AP-1 luciferase reporter plasmids were trypsinized, and 8 × 103 viable cells suspended in 100 μL of 5% FBS/MEM were added to each well of a 96-well plate and incubated at 37 °C in a humidified atmosphere of 5% CO2 . .. When the cells reached 80%–90% confluence, they were starved by culturing in 0.1% FBS MEM for an additional 24 h. The cells were then treated for 1 h with LicoA (0–10 μM) or Gc (0–10 μM) and irradiated with sUV for 6 h. After treatment, the cells were disrupted with 100 μL of lysis buffer (0.1 M potassium phosphate buffer (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol (DTT), and 2 mM EDTA), and the level of luciferase activity was measured using a luminometer (Luminoskan Ascent; Thermo Electron, Helsinki, Finland).

    Labeling:

    Article Title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study
    Article Snippet: The other materials are horseradish peroxidase (HRP) conjugated anti‑mouse IgG2b (Jackson ImmunoResearch, 115-035-207); 3,3′,5,5′-tetramethylbenzidine (TMB) (Kirkegaard & Perry Laboratories, 50-76-03); illustra™ Nap-10 columns (GE Healthcare, 17-0854-01); bovine serum albumin (BSA) (Equitech-Bio Inc.., BAH-1000) for the ELISA and (Sigma, 0547) for the LC-MS/MS assay; CHAPS (Research Organics, C3023–100G); ProClin 300 (Supelco, 48914-U); streptavidin (SA)-coated microplates (StreptaWell High bind 96 well) (Roche Diagnostics, 11989685001); stable isotope labeled peptide (Midwest Bio-Tech; customized order). .. Formic acid (94318) was purchased from Fluka; Trizma® hydrochloride (15506-017), iodoacetiamide (D-6003), dithiothreitol-DL (D-1532), Tween 20 (00–3005), trifluoroethanol (AM9856), sodium chloride (AM9760G) and EDTA (AM9261) were obtained from Ambion.

    Article Title: SNIP1 Recruits TET2 to Regulate c-MYC Target Genes and Cellular DNA Damage Response
    Article Snippet: 10 mM Dithiothreitol (DTT) was added and incubated for 30 minutes at 60°C, followed by addition of methyl methanethiosulfonate (MMTS) (ThermoFisher Scientific, Waltham, MA) to 20 mM. .. Digested peptides were labeled with 4-plex iTRAQ reagents (AB Sciex, Framingham, MA).

    Purification:

    Article Title: Genetic diversity and drug resistance of HIV-1 among infected pregnant women newly diagnosed in Luanda, Angola
    Article Snippet: The mix contained 25mM DNTP mix, 5X M-MLV buffer, 10mM of dithiothreitol (DTT), 40U of RNase OUT™ (Life Technologies, USA), 0.1mM of MMRTR6 primer ( 5’-TTTTACATCATTAGTGTGGG-3’ ), and 200U of M-MLV enzyme (Life Technologies, USA) [ ]. .. The amplicons were purified using the NZYGelpure Kit (Nzytech, Portugal), and sequenced using the ABI BigDye Terminator v3.1 reaction kit (Applied Biosystems, USA).

    Article Title: Salmonella Biofilm Development Depends on the Phosphorylation Status of RcsB
    Article Snippet: After 5 min of incubation at 65°C, samples were chilled on ice at least during 1 min, and a reverse transcription (RT) mix containing 44 μl of 5× first-strand buffer (Invitrogen), 22 μl of dithiothreitol (DTT) (0.1 M; Invitrogen), 2 μl of SuperScript III Retrotranscriptase (200 U/μl; Invitrogen), and 1 μl of RNase Out (40 U/μl; Invitrogen) was added to each preparation. cDNA was obtained after a cycle of 10 min at 25°C, 50 min at 50°C, and 5 min at 85°C. .. All preparations were purified by using CentriSep spin columns (Princeton separations).

    Polymerase Chain Reaction:

    Article Title: Genetic diversity and drug resistance of HIV-1 among infected pregnant women newly diagnosed in Luanda, Angola
    Article Snippet: Paragraph title: RNA extraction, cDNA synthesis, PCR and sequencing ... The mix contained 25mM DNTP mix, 5X M-MLV buffer, 10mM of dithiothreitol (DTT), 40U of RNase OUT™ (Life Technologies, USA), 0.1mM of MMRTR6 primer ( 5’-TTTTACATCATTAGTGTGGG-3’ ), and 200U of M-MLV enzyme (Life Technologies, USA) [ ].

    Article Title: 5-aza-2′-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells
    Article Snippet: Each reaction consisted of 1 μg of total RNA, 4 μl of 5x RT buffer, 2 μl of 0.1 M dithiothreitol (DTT), 0.5 μl of 0.5 μg/μl oligo(dT) primers (Invitrogen), 0.5 μl of 0.5 μg/μl single-stranded random hexanucleotides (Bioron), 1 μl of 10 mM dNTPs (Invitrogen) and 0.5 μl of SuperScript® II Reverse Transcriptase (200 U/μl), and was incubated at 37°C for 15 minutes, at 42°C for 60 minutes and at 90°C for 5 minutes. .. Primers used for PCR amplification are listed in Table .

    Lysis:

    Article Title: In-depth Analysis of the Magnaporthe oryzae Conidial Proteome
    Article Snippet: Lysis buffer (100 μl per 2 million conidia) containing 2M urea, 1X phosphate buffered saline and 0.1% SDS was used to extract the proteins by bead beating with 100 mg of 0.5 mm Zirconia/Silica beads (BioSpec Products, Inc., Bartlesville, OK) followed by boiling for 5 min. Debris and beads were spun down by centrifugation at 10,000 × g for 10 min at 4°C. .. Samples were dried down to 27 μl and 3 μl of 50 mM Dithiothreitol (DTT) (Thermo Fischer Scientific, Rockford, IL) was added to a final DTT concentration of 5 mM.

    Article Title: Licochalcone A, a Polyphenol Present in Licorice, Suppresses UV-Induced COX-2 Expression by Targeting PI3K, MEK1, and B-Raf
    Article Snippet: .. When the cells reached 80%–90% confluence, they were starved by culturing in 0.1% FBS MEM for an additional 24 h. The cells were then treated for 1 h with LicoA (0–10 μM) or Gc (0–10 μM) and irradiated with sUV for 6 h. After treatment, the cells were disrupted with 100 μL of lysis buffer (0.1 M potassium phosphate buffer (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol (DTT), and 2 mM EDTA), and the level of luciferase activity was measured using a luminometer (Luminoskan Ascent; Thermo Electron, Helsinki, Finland). .. PI3K Assay Active PI3K protein (100 ng) was incubated with LicoA or LY294002 at the indicated concentrations for 10 min at 30 °C.

    Nested PCR:

    Article Title: Genetic diversity and drug resistance of HIV-1 among infected pregnant women newly diagnosed in Luanda, Angola
    Article Snippet: The mix contained 25mM DNTP mix, 5X M-MLV buffer, 10mM of dithiothreitol (DTT), 40U of RNase OUT™ (Life Technologies, USA), 0.1mM of MMRTR6 primer ( 5’-TTTTACATCATTAGTGTGGG-3’ ), and 200U of M-MLV enzyme (Life Technologies, USA) [ ]. .. The obtained cDNA was subjected to a nested-PCR, targeting the protease (PR) and reverse transcriptase (RT) fragments of the HIV-1 pol gene, with an expected size of 1302 bp, using the protocol previously described [ ].

    Software:

    Article Title: Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile
    Article Snippet: cDNA was synthesized from 2.5 μg of total RNA by adding 1 μg N6 random primer, 10 mM deoxynucleoside triphosphates, 5× buffer, 100 mM dithiothreitol (DTT), 40 U RNase inhibitor (Life Technologies, Burlington, ON, Canada) in a total volume of 50 μl. .. The primers used to examine PaLoc gene transcripts were either synthesized using previously published sequences (for tcdA and tcdB [46]) or designed using Beacon Designer (version 4) software (Premier Biosoft, Palo Alto, CA) (for tcdE , tcdR , tcdC , fliA ) and synthesized (see Table S1 in the supplemental material).

    SYBR Green Assay:

    Article Title: 5-aza-2′-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells
    Article Snippet: Each reaction consisted of 1 μg of total RNA, 4 μl of 5x RT buffer, 2 μl of 0.1 M dithiothreitol (DTT), 0.5 μl of 0.5 μg/μl oligo(dT) primers (Invitrogen), 0.5 μl of 0.5 μg/μl single-stranded random hexanucleotides (Bioron), 1 μl of 10 mM dNTPs (Invitrogen) and 0.5 μl of SuperScript® II Reverse Transcriptase (200 U/μl), and was incubated at 37°C for 15 minutes, at 42°C for 60 minutes and at 90°C for 5 minutes. .. Quantitative PCR (qPCR) was performed utilizing the Applied Biosystems StepOne™ Real-Time PCR system using Absolute qPCR SYBR Green ROX Mix (Thermo Scientific).

    RNA Extraction:

    Article Title: Genetic diversity and drug resistance of HIV-1 among infected pregnant women newly diagnosed in Luanda, Angola
    Article Snippet: Paragraph title: RNA extraction, cDNA synthesis, PCR and sequencing ... The mix contained 25mM DNTP mix, 5X M-MLV buffer, 10mM of dithiothreitol (DTT), 40U of RNase OUT™ (Life Technologies, USA), 0.1mM of MMRTR6 primer ( 5’-TTTTACATCATTAGTGTGGG-3’ ), and 200U of M-MLV enzyme (Life Technologies, USA) [ ].

    Article Title: 5-aza-2′-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells
    Article Snippet: E6*I and E7 expression analysis, methylation-specific qPCR and miR-375 detection RNA extraction from cell lines was performed using the RNeasy Mini Kit (Qiagen) including DNaseI (Invitrogen) treatment according to the manufacturer`s instructions and the concentration was spectrophotometrically assessed by measuring the absorbance at A260/280 (NanoDrop 1000). .. Each reaction consisted of 1 μg of total RNA, 4 μl of 5x RT buffer, 2 μl of 0.1 M dithiothreitol (DTT), 0.5 μl of 0.5 μg/μl oligo(dT) primers (Invitrogen), 0.5 μl of 0.5 μg/μl single-stranded random hexanucleotides (Bioron), 1 μl of 10 mM dNTPs (Invitrogen) and 0.5 μl of SuperScript® II Reverse Transcriptase (200 U/μl), and was incubated at 37°C for 15 minutes, at 42°C for 60 minutes and at 90°C for 5 minutes.

    Sample Prep:

    Article Title: In-depth Analysis of the Magnaporthe oryzae Conidial Proteome
    Article Snippet: Paragraph title: 2.1 Sample preparation and digestion ... Samples were dried down to 27 μl and 3 μl of 50 mM Dithiothreitol (DTT) (Thermo Fischer Scientific, Rockford, IL) was added to a final DTT concentration of 5 mM.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study
    Article Snippet: The other materials are horseradish peroxidase (HRP) conjugated anti‑mouse IgG2b (Jackson ImmunoResearch, 115-035-207); 3,3′,5,5′-tetramethylbenzidine (TMB) (Kirkegaard & Perry Laboratories, 50-76-03); illustra™ Nap-10 columns (GE Healthcare, 17-0854-01); bovine serum albumin (BSA) (Equitech-Bio Inc.., BAH-1000) for the ELISA and (Sigma, 0547) for the LC-MS/MS assay; CHAPS (Research Organics, C3023–100G); ProClin 300 (Supelco, 48914-U); streptavidin (SA)-coated microplates (StreptaWell High bind 96 well) (Roche Diagnostics, 11989685001); stable isotope labeled peptide (Midwest Bio-Tech; customized order). .. Formic acid (94318) was purchased from Fluka; Trizma® hydrochloride (15506-017), iodoacetiamide (D-6003), dithiothreitol-DL (D-1532), Tween 20 (00–3005), trifluoroethanol (AM9856), sodium chloride (AM9760G) and EDTA (AM9261) were obtained from Ambion.

    Quantitation Assay:

    Article Title: SNIP1 Recruits TET2 to Regulate c-MYC Target Genes and Cellular DNA Damage Response
    Article Snippet: iTRAQ-based quantitative mass spectrometry To search for SNIP1-interacting transcription factors, multiplexed isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis was performed as described previously ( ). .. 10 mM Dithiothreitol (DTT) was added and incubated for 30 minutes at 60°C, followed by addition of methyl methanethiosulfonate (MMTS) (ThermoFisher Scientific, Waltham, MA) to 20 mM.

    Irradiation:

    Article Title: Licochalcone A, a Polyphenol Present in Licorice, Suppresses UV-Induced COX-2 Expression by Targeting PI3K, MEK1, and B-Raf
    Article Snippet: .. When the cells reached 80%–90% confluence, they were starved by culturing in 0.1% FBS MEM for an additional 24 h. The cells were then treated for 1 h with LicoA (0–10 μM) or Gc (0–10 μM) and irradiated with sUV for 6 h. After treatment, the cells were disrupted with 100 μL of lysis buffer (0.1 M potassium phosphate buffer (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol (DTT), and 2 mM EDTA), and the level of luciferase activity was measured using a luminometer (Luminoskan Ascent; Thermo Electron, Helsinki, Finland). .. PI3K Assay Active PI3K protein (100 ng) was incubated with LicoA or LY294002 at the indicated concentrations for 10 min at 30 °C.

    Acid Assay:

    Article Title: In-depth Analysis of the Magnaporthe oryzae Conidial Proteome
    Article Snippet: Protein concentrations were determined by Bicinchoninic acid assay. .. Samples were dried down to 27 μl and 3 μl of 50 mM Dithiothreitol (DTT) (Thermo Fischer Scientific, Rockford, IL) was added to a final DTT concentration of 5 mM.

    Concentration Assay:

    Article Title: In-depth Analysis of the Magnaporthe oryzae Conidial Proteome
    Article Snippet: .. Samples were dried down to 27 μl and 3 μl of 50 mM Dithiothreitol (DTT) (Thermo Fischer Scientific, Rockford, IL) was added to a final DTT concentration of 5 mM. .. Incubation for 30 min at 56 °C followed to reduce the protein disulfide bonds.

    Article Title: Salmonella Biofilm Development Depends on the Phosphorylation Status of RcsB
    Article Snippet: After 5 min of incubation at 65°C, samples were chilled on ice at least during 1 min, and a reverse transcription (RT) mix containing 44 μl of 5× first-strand buffer (Invitrogen), 22 μl of dithiothreitol (DTT) (0.1 M; Invitrogen), 2 μl of SuperScript III Retrotranscriptase (200 U/μl; Invitrogen), and 1 μl of RNase Out (40 U/μl; Invitrogen) was added to each preparation. cDNA was obtained after a cycle of 10 min at 25°C, 50 min at 50°C, and 5 min at 85°C. .. The cDNA quantity was measured, and the concentration was adjusted to 100 ng/μl.

    Article Title: 5-aza-2′-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells
    Article Snippet: E6*I and E7 expression analysis, methylation-specific qPCR and miR-375 detection RNA extraction from cell lines was performed using the RNeasy Mini Kit (Qiagen) including DNaseI (Invitrogen) treatment according to the manufacturer`s instructions and the concentration was spectrophotometrically assessed by measuring the absorbance at A260/280 (NanoDrop 1000). .. Each reaction consisted of 1 μg of total RNA, 4 μl of 5x RT buffer, 2 μl of 0.1 M dithiothreitol (DTT), 0.5 μl of 0.5 μg/μl oligo(dT) primers (Invitrogen), 0.5 μl of 0.5 μg/μl single-stranded random hexanucleotides (Bioron), 1 μl of 10 mM dNTPs (Invitrogen) and 0.5 μl of SuperScript® II Reverse Transcriptase (200 U/μl), and was incubated at 37°C for 15 minutes, at 42°C for 60 minutes and at 90°C for 5 minutes.

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Identification and characterization of an IgG sequence variant with an 11 kDa heavy chain C-terminal extension using a combination of mass spectrometry and high-throughput sequencing analysis
    Article Snippet: For chemicals used to make the LC-MS mobile phases, water and acetonitrile were purchased from Honeywell (Thermo Fisher scientific); formic acid (FA) from Pierce Thermo Fisher Scientific (Cat# 28905) and trifluoroacetic acid (TFA) from Sigma-Aldrich (Cat# T6508-10AMP). .. Dithiothreitol (DTT) was obtained from Thermo Fisher Scientific (Cat# A39255).

    Marker:

    Article Title: Separate mechanisms act concurrently to shed and release the prion protein from the cell
    Article Snippet: Peptide: N-glycosidase F (PNGase F) and prestained protein marker (broad range 7–175 kDa) were from New England BioLabs. .. Dithiothreitol (DTT) came from USB Corporation.

    Staining:

    Article Title: IL-17A-associated IKK-α signaling induced TSLP production in epithelial cells of COPD patients
    Article Snippet: In brief, after the collection of the sputum, the selected plugs were processed with 4 × w/v of 0.1% dithiothreitol (DTT), with the subsequent addition of 4 × w/v phosphate-buffered saline (PBS) (PBS 1 × ; Gibco). .. The cells obtained from IS were then cytocentrifuged (Cytospin 2; Shandon, Runcorn, UK) and stained with May–Grunwald–Giemsa.

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  • 90
    Thermo Fisher dtt
    Dynamic range of <t>roGFP2-Orp1</t> and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM <t>DTT</t> at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p
    Dtt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2501 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher m dithiothreitol
    Redox potential of the MetAP2 disulfide bond. A , the labeling of fully reduced MetAP2 by the biotin-linked maleimide, MPB, was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (DTT red ) to oxidized (DTT ox ) <t>dithiothreitol.</t> Biotin incorporation was measured by SDS-PAGE and blotting with streptavidin-peroxidase. B , plot of the ratio of reduced to oxidized MetAP2 as a function of the ratio of reduced to oxidized DTT. The solid line . The calculated equilibrium constant is 0.028 (95% confidence interval: 0.012–0.044). From the Nernst equation, the standard redox potential of the Cys 228 -Cys 448 disulfide is −261 mV. Data points and error bars , mean ± S.E. of three independent experiments. C , the initial rate of hydrolysis of Met-Gly-Pro-AMC by fully oxidized or reduced MetAP2 was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (Trx red ) to oxidized (Trx ox ) thioredoxin. The solid line . The calculated equilibrium constant is 0.740 (95% confidence interval: 0.600–0.688), which equates to a standard redox potential of −266 mV for the Cys 228 -Cys 448 disulfide bond. Data points and error bars , mean ± S.E. of three independent experiments.
    M Dithiothreitol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Test 12 samples for Rotaviurs Coronavirus E coli and Cryptosporidium Two well Antigen capture ELISA kits enable sensitive and specific detection of pathogens in animal samples allowing for reliable and
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    Dynamic range of roGFP2-Orp1 and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM DTT at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p

    Journal: PLoS ONE

    Article Title: H2O2 dynamics in the malaria parasite Plasmodium falciparum

    doi: 10.1371/journal.pone.0174837

    Figure Lengend Snippet: Dynamic range of roGFP2-Orp1 and HyPer-3 in transfected parasites within intact RBCs and after host cell lysis. After 15 s baseline monitoring, 3D7 parasites with intact or lysed host cells and transfected with roGFP2-Orp1 ( A ) or HyPer-3 ( C ) were exposed to 1 mM DIA and monitored for 2 min before adding 10 mM DTT at the CLSM. The fluorescence ratios (405/488 nm, 3D7 [roGFP2-Orp1] and 488/405 nm, 3D7 [HyPer-3] ) ( A, C ) at different time points are plotted against time. 3D7 [HyPer-3] ( C ) showed a higher DIA sensitivity than 3D7 [roGFP2-Orp1] ( A ) in both parasites residing in intact RBCs and those deprived of their host cell. Data from at least three trophozoites in three independent experiments were analyzed per data point. For measuring the dynamic range of both redox sensors in the parasites, the 405/488 nm ratio (3D7 [roGFP2-Orp1] ) ( B ) and the 488/405 nm ratio (3D7 [HyPer-3] ) ( D ) of fully oxidized and reduced probes were computed. The basal ratio, the ratio for 1 mM DIA, and 10 mM DTT after 2 min incubation (n > 27) of 3 independent experiments are shown. 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) with intact RBCs exhibited dynamic ranges of 5 and 7.3, respectively. The dynamic ranges for parasites after RBC lysis of 3D7 [roGFP2-Orp1] ( B ) and 3D7 [HyPer-3] ( D ) were 5.5 and 12.6, respectively. Mean values and standard error of the mean (SEM) are shown for all experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (*, p

    Article Snippet: Purified recombinant roGFP2-Orp1 and HyPer-3/SypHer proteins were reduced with 5 mM DTT for 10 min and 20 mM DTT for 30 min, respectively, at 4°C, desalinated (ZebaTM Spin Desalting Columns, Thermo Scientific), and diluted in reaction buffer to a final concentration of 5 μ M. A 5-fold drug/redox-active compound dilution (25 μ l) was mixed with 100 μ l of 5 μ M roGFP2-Orp1/HyPer in a 96-well microplate (black, half-area, Greiner Bio-One, Frickenhausen).

    Techniques: Transfection, Lysis, Confocal Laser Scanning Microscopy, Fluorescence, Incubation

    Dynamic range of roGFP2-Orp1 and Mito-roGFP2-Orp1 in transfected NF54- attB parasites. NF54- attB parasites transfected with roGFP2-Orp1 or Mito-roGFP2-Orp1 were exposed to 1 mM DIA or 10 mM DTT for 2 min before blocking with 2 mM NEM. Fluorescence ratios of 405/488 nm were detected with CLSM. NF54 [roGFP2-Orp1] - attB parasites showed a slightly higher DIA sensitivity than NF54 [Mito-roGFP2-Orp1] - attB parasites. CLSM data were composed of values from at least 10–20 trophozoites analyzed per experiment. Mean values and standard errors of the means (±SEM) are shown for three independent experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (***p

    Journal: Scientific Reports

    Article Title: Hydrogen peroxide dynamics in subcellular compartments of malaria parasites using genetically encoded redox probes

    doi: 10.1038/s41598-017-10093-8

    Figure Lengend Snippet: Dynamic range of roGFP2-Orp1 and Mito-roGFP2-Orp1 in transfected NF54- attB parasites. NF54- attB parasites transfected with roGFP2-Orp1 or Mito-roGFP2-Orp1 were exposed to 1 mM DIA or 10 mM DTT for 2 min before blocking with 2 mM NEM. Fluorescence ratios of 405/488 nm were detected with CLSM. NF54 [roGFP2-Orp1] - attB parasites showed a slightly higher DIA sensitivity than NF54 [Mito-roGFP2-Orp1] - attB parasites. CLSM data were composed of values from at least 10–20 trophozoites analyzed per experiment. Mean values and standard errors of the means (±SEM) are shown for three independent experiments. A one-way ANOVA test with 95% confidence intervals with the Dunnett’s Multiple Comparison Test was applied for statistical analysis of significance (***p

    Article Snippet: Purified recombinant roGFP2-Orp1 protein was reduced with 20 mM DTT for 30 min at 4 °C, desalinated (ZebaTM Spin Desalting Columns, Thermo Scientific), and diluted in reaction buffer to a final concentration of 5 µM.

    Techniques: Transfection, Blocking Assay, Fluorescence, Confocal Laser Scanning Microscopy

    BQ-induced enzyme inactivation and covalent modification of purified Trx1 protein. (A) Concentration-dependent inhibition of Trx1 activity by BQ. DTT-reduced Trx1 (2.5 μM) was incubated with BQ (0.5–10 μM) in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), with Trx1 activity determined after 5 min of treatment. (B) BQ-induced quinoprotein detection. DTT-reduced Trx1 (10 μM) was incubated with BQ (5–40 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed by SDS-PAGE followed by NBT redox staining. (C) Free thiol detection using BEI labelling. DTT-reduced Trx1 (5 μM) was incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by incubation with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection. * p

    Journal: Redox Biology

    Article Title: Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

    doi: 10.1016/j.redox.2019.101400

    Figure Lengend Snippet: BQ-induced enzyme inactivation and covalent modification of purified Trx1 protein. (A) Concentration-dependent inhibition of Trx1 activity by BQ. DTT-reduced Trx1 (2.5 μM) was incubated with BQ (0.5–10 μM) in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), with Trx1 activity determined after 5 min of treatment. (B) BQ-induced quinoprotein detection. DTT-reduced Trx1 (10 μM) was incubated with BQ (5–40 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed by SDS-PAGE followed by NBT redox staining. (C) Free thiol detection using BEI labelling. DTT-reduced Trx1 (5 μM) was incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by incubation with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection. * p

    Article Snippet: 2.4 Assessment of Trx1 oligomerization Trx (human or E. coli ) was reduced using DTT as described above, then 5 μM protein was incubated with different concentrations of BQ (2–80 μM) for 5 min. Aliquots of samples were then removed and diluted 4-fold with loading buffer (LDS Sample Buffer (4X) from Thermo Fisher, containing lithium dodecyl sulfate at pH 8.5 with SERVA Blue G250 and phenol red) with or without DTT.

    Techniques: Modification, Purification, Concentration Assay, Inhibition, Activity Assay, Incubation, SDS Page, Staining

    Identification of BQ-modified Cys residues in Trx1. DTT-reduced Trx1 (10 μM) was incubated with BQ (50 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The BQ-treated Trx1 was then digested using Lys-C at 37 °C overnight and analysed by LC-MS/MS (seen Materials and methods for further details). (A) Peptide 1, (B) Peptide 2, (C) Peptide 3 (cf. Table 1 ).

    Journal: Redox Biology

    Article Title: Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

    doi: 10.1016/j.redox.2019.101400

    Figure Lengend Snippet: Identification of BQ-modified Cys residues in Trx1. DTT-reduced Trx1 (10 μM) was incubated with BQ (50 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The BQ-treated Trx1 was then digested using Lys-C at 37 °C overnight and analysed by LC-MS/MS (seen Materials and methods for further details). (A) Peptide 1, (B) Peptide 2, (C) Peptide 3 (cf. Table 1 ).

    Article Snippet: 2.4 Assessment of Trx1 oligomerization Trx (human or E. coli ) was reduced using DTT as described above, then 5 μM protein was incubated with different concentrations of BQ (2–80 μM) for 5 min. Aliquots of samples were then removed and diluted 4-fold with loading buffer (LDS Sample Buffer (4X) from Thermo Fisher, containing lithium dodecyl sulfate at pH 8.5 with SERVA Blue G250 and phenol red) with or without DTT.

    Techniques: Modification, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Effect of GSH on BQ-induced enzyme activity loss, protein cross-linking and modification. Panels A, C, E: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) in the presence of GSH (5–1000 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The samples were split into three portions. The remaining Trx activity was measured (panel A), samples were analysed by SDS-PAGE followed by silver staining (panel C), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel E). Panels B, D, F: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), then GSH (5–1000 μM) was added and incubated for another 5 min. The samples were then split into three portions and analysed, as described above, for remaining Trx activity (panel B), by SDS-PAGE followed by silver staining (panel D), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel F). Data (mean ± standard deviations) from 3 independent experiments are presented in panels A and B. Representative images from 3 independent experiments are shown in panels (C - F). * p

    Journal: Redox Biology

    Article Title: Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

    doi: 10.1016/j.redox.2019.101400

    Figure Lengend Snippet: Effect of GSH on BQ-induced enzyme activity loss, protein cross-linking and modification. Panels A, C, E: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) in the presence of GSH (5–1000 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). The samples were split into three portions. The remaining Trx activity was measured (panel A), samples were analysed by SDS-PAGE followed by silver staining (panel C), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel E). Panels B, D, F: DTT-reduced Trx1 (2.5 μM) was incubated with BQ (20 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6), then GSH (5–1000 μM) was added and incubated for another 5 min. The samples were then split into three portions and analysed, as described above, for remaining Trx activity (panel B), by SDS-PAGE followed by silver staining (panel D), or incubated with BEI (250 μM) in dark for 30 min at 37 °C, then analysed by SDS-PAGE followed by treatment with horseradish peroxidase-conjugated streptavidin and enhanced chemiluminescence detection (panel F). Data (mean ± standard deviations) from 3 independent experiments are presented in panels A and B. Representative images from 3 independent experiments are shown in panels (C - F). * p

    Article Snippet: 2.4 Assessment of Trx1 oligomerization Trx (human or E. coli ) was reduced using DTT as described above, then 5 μM protein was incubated with different concentrations of BQ (2–80 μM) for 5 min. Aliquots of samples were then removed and diluted 4-fold with loading buffer (LDS Sample Buffer (4X) from Thermo Fisher, containing lithium dodecyl sulfate at pH 8.5 with SERVA Blue G250 and phenol red) with or without DTT.

    Techniques: Activity Assay, Modification, Incubation, SDS Page, Silver Staining

    BQ-induced protein cross-linking. Human Trx1 (5 μM, A and B) and E. coli Trx (5 μM, C and D), pre-treated with DTT, were incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed under non-reducing (A and C) and reducing (B and D) conditions, and then analysed by SDS-PAGE with silver staining. Representative images from 3 independent experiments are shown in each panel.

    Journal: Redox Biology

    Article Title: Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

    doi: 10.1016/j.redox.2019.101400

    Figure Lengend Snippet: BQ-induced protein cross-linking. Human Trx1 (5 μM, A and B) and E. coli Trx (5 μM, C and D), pre-treated with DTT, were incubated with BQ (2–80 μM) for 5 min in 50 mM Tris-HCl containing 2 mM EDTA (pH 7.6). Samples were analysed under non-reducing (A and C) and reducing (B and D) conditions, and then analysed by SDS-PAGE with silver staining. Representative images from 3 independent experiments are shown in each panel.

    Article Snippet: 2.4 Assessment of Trx1 oligomerization Trx (human or E. coli ) was reduced using DTT as described above, then 5 μM protein was incubated with different concentrations of BQ (2–80 μM) for 5 min. Aliquots of samples were then removed and diluted 4-fold with loading buffer (LDS Sample Buffer (4X) from Thermo Fisher, containing lithium dodecyl sulfate at pH 8.5 with SERVA Blue G250 and phenol red) with or without DTT.

    Techniques: Incubation, SDS Page, Silver Staining

    Redox potential of the MetAP2 disulfide bond. A , the labeling of fully reduced MetAP2 by the biotin-linked maleimide, MPB, was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (DTT red ) to oxidized (DTT ox ) dithiothreitol. Biotin incorporation was measured by SDS-PAGE and blotting with streptavidin-peroxidase. B , plot of the ratio of reduced to oxidized MetAP2 as a function of the ratio of reduced to oxidized DTT. The solid line . The calculated equilibrium constant is 0.028 (95% confidence interval: 0.012–0.044). From the Nernst equation, the standard redox potential of the Cys 228 -Cys 448 disulfide is −261 mV. Data points and error bars , mean ± S.E. of three independent experiments. C , the initial rate of hydrolysis of Met-Gly-Pro-AMC by fully oxidized or reduced MetAP2 was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (Trx red ) to oxidized (Trx ox ) thioredoxin. The solid line . The calculated equilibrium constant is 0.740 (95% confidence interval: 0.600–0.688), which equates to a standard redox potential of −266 mV for the Cys 228 -Cys 448 disulfide bond. Data points and error bars , mean ± S.E. of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Redox Regulation of Methionine Aminopeptidase 2 Activity *

    doi: 10.1074/jbc.M114.554253

    Figure Lengend Snippet: Redox potential of the MetAP2 disulfide bond. A , the labeling of fully reduced MetAP2 by the biotin-linked maleimide, MPB, was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (DTT red ) to oxidized (DTT ox ) dithiothreitol. Biotin incorporation was measured by SDS-PAGE and blotting with streptavidin-peroxidase. B , plot of the ratio of reduced to oxidized MetAP2 as a function of the ratio of reduced to oxidized DTT. The solid line . The calculated equilibrium constant is 0.028 (95% confidence interval: 0.012–0.044). From the Nernst equation, the standard redox potential of the Cys 228 -Cys 448 disulfide is −261 mV. Data points and error bars , mean ± S.E. of three independent experiments. C , the initial rate of hydrolysis of Met-Gly-Pro-AMC by fully oxidized or reduced MetAP2 was used to calculate the ratio of reduced to oxidized enzyme as a function of the ratio of reduced (Trx red ) to oxidized (Trx ox ) thioredoxin. The solid line . The calculated equilibrium constant is 0.740 (95% confidence interval: 0.600–0.688), which equates to a standard redox potential of −266 mV for the Cys 228 -Cys 448 disulfide bond. Data points and error bars , mean ± S.E. of three independent experiments.

    Article Snippet: Just before use, the active site disulfide bond of the oxidoreductase was reduced with 50 m m dithiothreitol and 50 m m tris-(2-carboxyethyl)phosphine (TCEP) for 1 h at 25 °C, and the reducing agents were removed by two passes through Zeba spin desalting columns equilibrated with phosphate-buffered saline (Thermo Scientific).

    Techniques: Labeling, SDS Page