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dtt-dithiothreitol

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Identification of TBC1D4 interacting partners in human skeletal muscle. Exp...
Published: 22 February 2022
Figure 1 Identification of TBC1D4 interacting partners in human skeletal muscle. Experimental approach for mapping the TBC1D4 interactome in human skeletal muscle before and after (b/a) a hyperinsulinemic-euglycemic clamp (HEC) (A) and in WT and TBC1D4 whole-body KO mouse skeletal muscle at basal (B). DTT, dithiothreitol. C: Principal component analysis plot including principal components 1 and 2 on all quantified proteins in human (left) and mouse (right) muscle. Dots represent individual samples, and colored circles are manually inserted. Volcano plots based on the Student t test for the human (D) and mouse TBC1D4 interactome (E) with log2-fold change difference (human: TBC1D4 IP – IgG IP or mouse: WT-TBC1D4 KO) on the x-axis and –log10P values on the y-axis. Significant interactors are marked in blue. Owing to low abundance and exclusive quantification of some interactors, imputation of missing values in the corresponding control samples can make a significant interactor appear with a negative log2 difference. F: Proteins in which their interaction with TBC1D4 is regulated by insulin stimulation either by Student t test (bar graph with mean ± SEM) or exclusive identification ( Table ). G: Venn diagram illustrating significant TBC1D4 interactors found in human, mouse, or both species. Detailed information on identification of interactors found in both human and mouse muscle. More
Journal Articles
Journal: Diabetes
Diabetes 1981;30(8):694–699
Published: 01 August 1981
...). In each of the diabetic groups (48, 72, and 96 h), the mean enzyme activity was significantly less (P < 0.005) than the control mean. Homogenate enrichment with the thiol reagent dithiothreitol (DTT) (5 mM) failed to reverse this diabetic effect. Moreover, although the hepatic content of NP-SH groups...
Journal Articles
Journal: Diabetes
Diabetes 1979;28(9):810–817
Published: 01 September 1979
... degradation by membranes, whereas glutathione (GSH) and dithíothreitol (DTT) were stimulatory. These characteristics of insulin-degrading activity in the plasma membrane (membrane-IDE) were similar to those of the partially purified insulin-degrading enzyme from the cytosol fraction (cytosol-IDE). To clarify...
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Basal level of processed ATF6 is elevated in <em>Perk</em>-deficien...
Published: 08 June 2010
FIG. 4. Basal level of processed ATF6 is elevated in Perk-deficient cells. AD293 cells were cotransfected with EYFP-ATF6α and either an empty vector (Vector) or DNPerk. Empty-vector cotransfected samples were either left untreated, while samples treated with 5 mmol/l dithiothreitol (DTT) or 5 μg/ml brefeldin A (BFA) for 1 h served as controls for ATF6 induction. Immunoblotting with an antibody that recognizes YFP-tagged ATF6 shows both the ER-resident uncleaved (fl, full length) and cleaved nuclear (n) forms. Quantification of the nuclear ATF6 expressed as a percentage of the full-length form is shown below the tubulin panel. FIG. 4. Basal level of processed ATF6 is elevated in Perk-deficient cells. AD293 cells were cotransfected with EYFP-ATF6α and either an empty vector (Vector) or DNPerk. Empty-vector cotransfected samples were either left untreated, while samples treated with 5 mmol/l dithiothreitol (DTT) or 5 μg/ml brefeldin A (BFA) for 1 h served as controls for ATF6 induction. Immunoblotting with an antibody that recognizes YFP-tagged ATF6 shows both the ER-resident uncleaved (fl, full length) and cleaved nuclear (n) forms. Quantification of the nuclear ATF6 expressed as a percentage of the full-length form is shown below the tubulin panel. More
Journal Articles
Journal: Diabetes
Diabetes 2004;53(6):1599–1602
Published: 01 June 2004
... in receptor binding is likely to require a conformational change to expose a hidden functional surface. Our results demonstrate that this surface spans both chains of the insulin molecule and includes sites of rare human mutations that cause diabetes. DTT, dithiothreitol FnIII1, second...
Journal Articles
Journal: Diabetes
Diabetes 1997;46(3):401–407
Published: 01 March 1997
..., the relative effectiveness of the two receptors was assessed. The α2adrenergic receptor was found to be markedly more effective than the galanin receptor in activating G-proteins. 1 BSA, bovine serum albumin; CTX, cholera toxin; DTT, dithiothreitol; KATP, ATP-sensitive potassium...
Journal Articles
Journal: Diabetes
Diabetes 1996;45(Supplement_3):S117–S119
Published: 01 July 1996
... previously (3). Partial purification and assay of PKC Accepted for publication 26 September 1995. ASMC, aortic smooth muscle cell; DAG, diacylglycerol; DMEM, Dulbecco's Cells. Confluent ASMCs were washed twice with phosphate-buffered modified Eagle's medium; DTT, dithiothreitol; PKC, protein kinase C; PMA...
Journal Articles
Journal: Diabetes
Diabetes 2003;52(4):926–928
Published: 01 April 2003
... January 2003. AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; AMPK, AMP-activated protein kinase; CreaT, creatine transporter; DTT, dithiothreitol; H1, histone 1; HKII, hexokinase II; L-PK, liver-type pyruvate kinase; MEF2, myocyte enhancer factor 2; NRF-1, nuclear respiratory factor 1. All...
Journal Articles
Journal: Diabetes
Diabetes 1997;46(6):929–936
Published: 01 June 1997
... Medicine, Kumamoto University School of Medicine, 1- 1-1, Honjo, Kumamoto, 860, Japan. 1 DTT, dithiothreitol; GRB2, growth factor receptor–bound protein 2; IRS-1, insulin receptor substrate-1; MAP, mitogen-activated protein; p85, 85-kDa regulatory subunit of PI 3-kinase; PI 3-kinase...
Meeting Abstracts
Journal: Diabetes
Diabetes 1998;47(7):1066–1073
Published: 01 July 1998
... of From the Laboratory of Molecular Endocrinology, Howard Hughes Medical pancreatic -cells. We also report that cytosolic dithiothreitol Institute, Harvard Medical School, Massachusetts General Hospital, (DTT; a sulfhydryl-reducing agent), but not extracellular DTT, Boston, Massachusetts. inhibits...
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Stable LAL knockdown affects adipocyte cholesterol homeostasis. <em>A</em>...
Published: 02 November 2020
Figure 3 Stable LAL knockdown affects adipocyte cholesterol homeostasis. A: Stratification of stable transfectants according to Lipa residual expression. A total of 17 independent cell pools were analyzed for Lipa mRNA and assigned to one of the three groups by comparison with parental nontransfected controls. B: Quantitative RT-PCR determination of cholesterol-regulated genes’ mRNA in cell groups with gradual Lipa inhibition. Significant differences by Student t test are shown. C: Spearman correlation of Srebf2 mRNA and Lipa residual expression. Correlation coefficient is indicated; P < 0.05. D: Pretreatment of cells with exogenous free cholesterol to replenish intracellular pools was performed by adding 1 mg/mL cholesterol (catalog no. C3045; Sigma-Aldrich) in DMEM containing 1% of fatty acid free BSA for 1 h. Medium was removed and cells were incubated in fresh medium for the subsequent 4 h before lysis. Hmgcr and Srebf2 mRNAs were assessed as described. E: Perilipin fluorescence intensity normalized to cell area was quantified in Lipa-deficient cells and control cells. F: FAS protein content was assessed by Western blot in clones with different degrees of Lipa inhibition and normalized to β-actin. G: LD diameter measurement using PerfectImage Software (Claravision). Each point represents mean diameter values ± SEM of independent cell pools, plotted against Lipa residual expression. Statistically significant (P < 0.05) negative correlation linking LAL expression and LD size is shown with correlation coefficient. ER stress markers Grp78 (H) and Atf4 (I) mRNA were assessed after cell incubation for 6 h in serum-free medium before addition of 0.5 mmol/L Dithiothréitol (DTT) (catalog no. D1532, Invitrogen) for 1 h. Three independent experiments were performed, with six independent stable transformants in each group. Values are mean ± SEM, normalized to 18S RNA. Med, medium; rel, relative. Figure 3. Stable LAL knockdown affects adipocyte cholesterol homeostasis. A: Stratification of stable transfectants according to Lipa residual expression. A total of 17 independent cell pools were analyzed for Lipa mRNA and assigned to one of the three groups by comparison with parental nontransfected controls. B: Quantitative RT-PCR determination of cholesterol-regulated genes’ mRNA in cell groups with gradual Lipa inhibition. Significant differences by Student t test are shown. C: Spearman correlation of Srebf2 mRNA and Lipa residual expression. Correlation coefficient is indicated; P < 0.05. D: Pretreatment of cells with exogenous free cholesterol to replenish intracellular pools was performed by adding 1 mg/mL cholesterol (catalog no. C3045; Sigma-Aldrich) in DMEM containing 1% of fatty acid free BSA for 1 h. Medium was removed and cells were incubated in fresh medium for the subsequent 4 h before lysis. Hmgcr and Srebf2 mRNAs were assessed as described. E: Perilipin fluorescence intensity normalized to cell area was quantified in Lipa-deficient cells and control cells. F: FAS protein content was assessed by Western blot in clones with different degrees of Lipa inhibition and normalized to β-actin. G: LD diameter measurement using PerfectImage Software (Claravision). Each point represents mean diameter values ± SEM of independent cell pools, plotted against Lipa residual expression. Statistically significant (P < 0.05) negative correlation linking LAL expression and LD size is shown with correlation coefficient. ER stress markers Grp78 (H) and Atf4 (I) mRNA were assessed after cell incubation for 6 h in serum-free medium before addition of 0.5 mmol/L Dithiothréitol (DTT) (catalog no. D1532, Invitrogen) for 1 h. Three independent experiments were performed, with six independent stable transformants in each group. Values are mean ± SEM, normalized to 18S RNA. Med, medium; rel, relative. More
Meeting Abstracts
Journal: Diabetes
Diabetes 1998;47(12):1836–1840
Published: 01 December 1998
... protein kinase A (PKA). August 1998. Amino-acid sequence analysis reveals that human GFA has Q.K.H and E.A.G. hold stock in Monsanto. two potential PKA-phosphorylation sites, 202RRGS205 and DMEM, Dulbecco s modified Eagle s medium; DTT, dithiothreitol; FCS, 232KKGS235; one of these consensus sites...
Meeting Abstracts
Journal: Diabetes
Diabetes 1999;48(4):685–690
Published: 01 April 1999
... serum. Con uent 3T3-L1 preadipocytes were ing protein; CED, -cyanoethyl-N, N-diisopropylamino; DMEM, Dulbecco s induced to differentiation into adipocytes, as previously described (17). Brie y, modified Eagle s medium; DTT, dithiothreitol; FBS, fetal bovine serum; 1 day after con uence, cells were...
Journal Articles
Journal: Diabetes
Diabetes 1997;46(5):847–853
Published: 01 May 1997
... this hypothesis, we measured the activities of MEK cPLA2, cytosolic phospholipase A2; DTT, dithiothreitol; ECM, extracel- (MAPK or ERK kinase) and MAPK in diabetic glomeruli and lular matrix; ERK, extracellular signal-regulated kinase; FBS, fetal bovine mesangial cells cultured under high glucose conditions...
Meeting Abstracts
Journal: Diabetes
Diabetes 1998;47(10):1549–1554
Published: 01 October 1998
...@joslab.harvard.edu. RESEARCH DESIGN AND METHODS Received for publication 30 April 1998 and accepted in revised form 2 July 1998. Animals and experimental design In vivo studies. Sprague-Dawley rats weighing 150 200 g were purchased from DTT, dithiothreitol; ECL, enhanced chemiluminescence; ERK, extracel- Taconic...
Journal Articles
Journal: Diabetes
Diabetes 1983;32(9):798–803
Published: 01 September 1983
.... *The following abbreviations are used throughout this article: T4, L-thyroxine; Address reprint requests to Dr. Laurence A. Gavin, Endocrine-Metabolic Serv- T3, 3,5,3'-triiodothyronine, DTT, dithiothreitol; SRIF, somatostatin. ice (115), Veterans Administration Medical Center, 4150 Clement Street, San Francisco...
Journal Articles
Journal: Diabetes
Diabetes 1994;43(5):676–683
Published: 01 May 1994
... dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TSC, triple-stranded collagen; DTT, dithiothreitol; Tiron, 4,5-dihydroxy-l,3-benzenedisulfonic acid diso- dium salt. 676 DIABETES, VOL. 43, MAY 1994 FU AND ASSOCIATES collagen in vitro but did not evaluate the role...
Journal Articles
Journal: Diabetes
Diabetes 1990;39(1):31–37
Published: 01 January 1990
... M sodium phosphate at pH 7, pass both the a1(VI) and a2(VI) collagen subunits (20). The containing 20 mM dithiothreitol (DTT) (extract 2) in the pres- anti-type VI collagen serum also reacted with a smaller-mo- ence of protease inhibitors as previously reported (3). lecular-weight polypeptide {Mr...
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H<sub>2</sub>S S-sulfhydrylated Keap1 at Cys151 to regulate Nrf2 transcript...
Published: 22 June 2016
Figure 8 H2S S-sulfhydrylated Keap1 at Cys151 to regulate Nrf2 transcription activity and reduce the generation of ROS in HG+ox-LDL–treated endothelial cells. A: EA.hy926 endothelial cells were treated with GYY4137 (100 μmol/L) followed by d-glucose (D-Glu) (25 mmol/L) plus ox-LDL (50 mg/L) stimulation for 24 h. Cell lysates were immunoprecipitated with an anti-Keap1 or an anti-IgG antibody (negative control) and blotted with an anti-Nrf2 antibody (top panel). An aliquot of total lysate was analyzed for Keap1, Nrf2, and GAPDH expression (bottom panel). B: EA.hy926 endothelial cells were treated with dithiothreitol (DTT) (1 mmol/L, negative control) or d-glucose (25 mmol/L) plus ox-LDL (50 mg/L) in the presence or absence of GYY4137 (100 μmol/L) for 2 h. S-sulfhydration on Keap1 was detected with the “tag-switch” method. C: After plasmid transfection of Keap1-WT or mutated Keap1 at Cys151, Cys273, and Cys288 for 24 h followed by GYY4137 (100 μmol/L) treated for another 2 h, S-sulfhydration on Keap1 was detected with the “tag-switch” method. D: Transfected cells were treated with d-glucose (25 mmol/L) plus ox-LDL (50 mg/L) in the presence or absence of GYY4137 (100 μmol/L) for another 24 h, cell lysates were immunoprecipitated with an anti-Keap1 antibody, and the immunoprecipitated proteins were subjected to immunoblot analysis with anti-Nrf2 antibodies (top panel). The total lysates were analyzed with anti-Keap1, anti-Nrf2, and anti-GAPDH antibodies (bottom panel). E and F: Transfected cells were treated with d-glucose (25 mmol/L) plus ox-LDL (50 mg/L) in the presence or absence of GYY4137 (100 μmol/L) for 24 h. Nuclear extracts prepared from cells were subjected to Western blotting analysis for detecting the nuclear localization of Nrf2 (n = 4). ROS accumulation was determined by the DHE assay. Scale bars, 50 μm. Data shown are mean ± SEM. **P < 0.01 vs. Keap1-WT–transfected cells treated with d-glucose plus ox-LDL. G: Quantification of DHE fluorescence image of F. **P < 0.01 vs. untreated Keap1-WT–transfected cells; ##P < 0.01 vs. Keap1-WT–transfected cells treated with d-glucose and ox-LDL; &&P < 0.01 vs. untreated C151A-transfected cells. n = 4. Figure 8. H2S S-sulfhydrylated Keap1 at Cys151 to regulate Nrf2 transcription activity and reduce the generation of ROS in HG+ox-LDL–treated endothelial cells. A: EA.hy926 endothelial cells were treated with GYY4137 (100 μmol/L) followed by d-glucose (D-Glu) (25 mmol/L) plus ox-LDL (50 mg/L) stimulation for 24 h. Cell lysates were immunoprecipitated with an anti-Keap1 or an anti-IgG antibody (negative control) and blotted with an anti-Nrf2 antibody (top panel). An aliquot of total lysate was analyzed for Keap1, Nrf2, and GAPDH expression (bottom panel). B: EA.hy926 endothelial cells were treated with dithiothreitol (DTT) (1 mmol/L, negative control) or d-glucose (25 mmol/L) plus ox-LDL (50 mg/L) in the presence or absence of GYY4137 (100 μmol/L) for 2 h. S-sulfhydration on Keap1 was detected with the “tag-switch” method. C: After plasmid transfection of Keap1-WT or mutated Keap1 at Cys151, Cys273, and Cys288 for 24 h followed by GYY4137 (100 μmol/L) treated for another 2 h, S-sulfhydration on Keap1 was detected with the “tag-switch” method. D: Transfected cells were treated with d-glucose (25 mmol/L) plus ox-LDL (50 mg/L) in the presence or absence of GYY4137 (100 μmol/L) for another 24 h, cell lysates were immunoprecipitated with an anti-Keap1 antibody, and the immunoprecipitated proteins were subjected to immunoblot analysis with anti-Nrf2 antibodies (top panel). The total lysates were analyzed with anti-Keap1, anti-Nrf2, and anti-GAPDH antibodies (bottom panel). E and F: Transfected cells were treated with d-glucose (25 mmol/L) plus ox-LDL (50 mg/L) in the presence or absence of GYY4137 (100 μmol/L) for 24 h. Nuclear extracts prepared from cells were subjected to Western blotting analysis for detecting the nuclear localization of Nrf2 (n = 4). ROS accumulation was determined by the DHE assay. Scale bars, 50 μm. Data shown are mean ± SEM. **P < 0.01 vs. Keap1-WT–transfected cells treated with d-glucose plus ox-LDL. G: Quantification of DHE fluorescence image of F. **P < 0.01 vs. untreated Keap1-WT–transfected cells; ##P < 0.01 vs. Keap1-WT–transfected cells treated with d-glucose and ox-LDL; &&P < 0.01 vs. untreated C151A-transfected cells. n = 4. More
Images
Experimental diabetes alters the osmotic swelling characteristics of Mülle...
Published: 01 March 2006
FIG. 1. Experimental diabetes alters the osmotic swelling characteristics of Müller glial cells in the rat retina. Retinal slices from 6-month diabetic and age-matched untreated control animals were perfused with a hypotonic solution, and the cross-sectional area of glial cell somata in the inner nuclear layer was recorded. A: Exposure of the slices to hypotonic solution induced time-dependent soma swelling of glial cells in a diabetic retina but had no effect on the soma volume of cells in a control retina. However, glial cell bodies in control retinas display hypotonic swelling in the presence of Ba2+ (1 mmol/l) in the bathing solution. The images shown above display an original record of a dye-filled glial cell body in a retinal slice from a diabetic animal, dependent on the time of hypotonic exposure. Scale bar, 5 μm. B: Mean cross-sectional area of glial cell bodies in retinas from diabetic and control animals during hypotonic conditions, in the absence and presence of Ba2+ (1 mmol/l). C: The osmotic glial cell swelling in diabetic retinas was inhibited in the presence of the following agents: the selective inhibitor of PLA2 activation, 4-bromophenacyl bromide (bromo; 300 μmol/l); the cyclooxygenase inhibitor, indomethacin (indo; 10 μmol/l); the corticosteroid, triamcinolone acetonide (triam; 100 μmol/l); and the reducing agent, dithiothreitol (DTT; 3 mmol/l). D: Triamcinolone acetonide (triam; 100 μmol/l) inhibited the glial cell swelling in control retinas, which was evoked by hypotonic challenge in the presence of Ba2+ (1 mmol/l), arachidonic acid (AA; 10 μmol/l), prostaglandin E2 (PGE2; 30 nmol/l), or H2O2 (50 μmol/l). BD: Values were measured after a 4-min perfusion of the hypotonic solution and are expressed as percent of the control value measured before application of the solution (100%). The data were obtained from six diabetic and five control retinas. Cell numbers are within the bars. Significant difference vs. control (100%): **P < 0.01; ***P < 0.001. Significant difference between cells from control and diabetic retinas: ○○○P < 0.001. Significant blocking effects: ••P < 0.01; •••P < 0.001. FIG. 1. Experimental diabetes alters the osmotic swelling characteristics of Müller glial cells in the rat retina. Retinal slices from 6-month diabetic and age-matched untreated control animals were perfused with a hypotonic solution, and the cross-sectional area of glial cell somata in the inner nuclear layer was recorded. A: Exposure of the slices to hypotonic solution induced time-dependent soma swelling of glial cells in a diabetic retina but had no effect on the soma volume of cells in a control retina. However, glial cell bodies in control retinas display hypotonic swelling in the presence of Ba2+ (1 mmol/l) in the bathing solution. The images shown above display an original record of a dye-filled glial cell body in a retinal slice from a diabetic animal, dependent on the time of hypotonic exposure. Scale bar, 5 μm. B: Mean cross-sectional area of glial cell bodies in retinas from diabetic and control animals during hypotonic conditions, in the absence and presence of Ba2+ (1 mmol/l). C: The osmotic glial cell swelling in diabetic retinas was inhibited in the presence of the following agents: the selective inhibitor of PLA2 activation, 4-bromophenacyl bromide (bromo; 300 μmol/l); the cyclooxygenase inhibitor, indomethacin (indo; 10 μmol/l); the corticosteroid, triamcinolone acetonide (triam; 100 μmol/l); and the reducing agent, dithiothreitol (DTT; 3 mmol/l). D: Triamcinolone acetonide (triam; 100 μmol/l) inhibited the glial cell swelling in control retinas, which was evoked by hypotonic challenge in the presence of Ba2+ (1 mmol/l), arachidonic acid (AA; 10 μmol/l), prostaglandin E2 (PGE2; 30 nmol/l), or H2O2 (50 μmol/l). B–D: Values were measured after a 4-min perfusion of the hypotonic solution and are expressed as percent of the control value measured before application of the solution (100%). The data were obtained from six diabetic and five control retinas. Cell numbers are within the bars. Significant difference vs. control (100%): **P < 0.01; ***P < 0.001. Significant difference between cells from control and diabetic retinas: ○○○P < 0.001. Significant blocking effects: ••P < 0.01; •••P < 0.001. More