Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 8

1.
Figure 5

Figure 5. Sensitivity of TR3-knockdown cells to oxidative stress. From: Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

siTR3 1.1-knockdown cells or cells stably transfected with pSec hygro (vector) were treated with the indicated concentrations of oxidants, cell viability was determined, and the results represented as means ± S.D. of four replicates. (A) Treatment with hydrogen peroxide. (B) Treatment with menadione. (C) Treatment with diamide.

Anton A. Turanov, et al. Biochem J. 2010 September 1;430(2):285-293.
2.
Figure 3

Figure 3. Sensitivity of TR1-knockdown cells to oxidative stress. From: Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

TCMK-1 cells were stably transfected with pU6 (vector), pU6-TR1 overexpressing construct (TR1) and TR1-knockdown construct siTR1-3 (siTR1). (A) Levels of TR1 and TR3 expression in control, TR1-overexpressing and TR1-knockdown cells as assayed by Western blotting. (B) TCMK-1 control and TR1-knockdown cells were treated with the indicated concentrations of hydrogen peroxide, cell viability was determined and the results are represented as means ± S.D. of four replicates. (C) TCMK-1 control and TR1-knockdown cells were treated with the indicated concentrations of diamide, cell viability was determined and the results are represented as means ± S.D. of four replicates.

Anton A. Turanov, et al. Biochem J. 2010 September 1;430(2):285-293.
3.
Figure 8

Figure 8. Analysis of the redox state of Trxs in livers of WT and liver-specific selenocysteine tRNA-knockout mice. From: Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

Liver extracts were treated with AMS, a compound that alkylates cysteine residues resulting in a decreased mobility on gels. Control (WT) samples were incubated with diamide (DA) or with DTT prior to AMS alkylation to generate oxidized and reduced Trx samples respectively (two left-hand lanes). Proteins were subjected to SDS/PAGE followed by Western blot analysis with antibodies specific for Trx1.

Anton A. Turanov, et al. Biochem J. 2010 September 1;430(2):285-293.
4.
Figure 7

Figure 7. Analysis of Trx expression levels in mice with liver-specific knockout of selenocysteine tRNA. From: Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

Liver lysates of indicated trsp genotypes were subjected to ADP–Sepharose affinity isolation. Isolated proteins were analysed by SDS/PAGE followed by Western blotting with antibodies specific for Trx1 or Trx2. (A) Initial, flow-through and elution fractions from ADP–Sepharose were analysed. (B) Only the initial liver lysates were analysed. Initial, flow and elution indicate initial liver lysates which were loaded on to ADP–Sepharose, flow-through fractions containing unbound proteins and elution fractions enriched for TRs respectively. (C and D) Band intensities from the initial fractions for Trx1 and Trx2 correspond to WT, heterozygous and homozygous knockout samples (as shown in B, initial) were quantified using ImageQuant. Results are represented as means ± S.D. for three independent experiments in which values for WT samples correspond to 100 %.

Anton A. Turanov, et al. Biochem J. 2010 September 1;430(2):285-293.
5.
Figure 6

Figure 6. 75Se metabolic labelling and affinity isolation of TRs from mice with liver-specific Trsp knockout on ADP–Sepharose. From: Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

Mice with genotypes Trspfl/fll-Alb-Cre+/− (+/−), Trspfl/+-Alb-Cre+/− (−/−) and Trspfl/fll (WT) were labelled with 75Se. Liver lysates were loaded on to ADP–Sepharose resins, the resins extensively washed with loading buffer and bound proteins eluted with SDS/PAGE loading buffer. Isolated proteins were subjected to SDS/PAGE followed by Western blotting with antibodies specific for TR1, TR3, Trx1 and Trx2. (A) 75Se-labelled proteins detected with a PhosphorImager after affinity isolation of TRs. Molecular mass markers (in kDa) are shown on the left-hand side. (B) Western blotting analysis of TR1, TR3, Trx1 and Trx2 expression in samples shown in (A). Initial, flow and elution indicate initial liver lysates which were loaded on to ADP–Sepharose, flow-through fractions containing unbound proteins and elution fractions enriched for TRs respectively.

Anton A. Turanov, et al. Biochem J. 2010 September 1;430(2):285-293.
6.
Figure 2

Figure 2. Structural analysis of TR1–Trx1 interaction. From: Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

Docking of TR1 (in cyan, with violet ribbon representation) with Trx1 (in red and yellow ribbons) is shown. Catalytic cysteine residues (Cys32 and Cys35 for Trx1, and Cys497 and Sec498 for TR1) are shown in stick representation. The C-terminal active site of TR1 is shown in blue wire frame (except for the catalytic cysteine residue, depicted in stick representation). The Figure shows the best docking position found (energy score for the complex is −370 kcal/mol) from two different angles (45 °rotation in the direction of the arrow shown). Helices 1 and 3 are labelled. Helix 1 is the first helix in the TR1 structure (spans residues 22–34). Cys497 and Sec498 are close to helix 3 and helix 1 respectively. In our analysis, the interaction between Trx1 and TR1 occurs on the side of helix 3, bringing Cys32 in Trx1 close to Cys497.

Anton A. Turanov, et al. Biochem J. 2010 September 1;430(2):285-293.
7.
Figure 1

Figure 1. Identification of TR1 targets in cytosolic fractions of mouse and rat tissues. From: Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

Mouse liver, rat liver, rat brain cytosolic fractions, or mouse serum, were loaded on to the TR1-immobilized resins indicated (designated based on amino acids at the cysteine–selenocysteine positions in the C-terminus of TR1, see text for description). Bound proteins were eluted with DTT and analysed by SDS/PAGE. Coomassie Blue staining was used to visualize proteins. (A) Mouse liver cytosol, (B) rat liver cytosol and (C) rat brain cytosol. Lower panels (AC) show Western blotting with anti-Trx1 antibodies (mouse liver and rat liver cytosolic fractions were used as controls for rat brain). (D) Mouse serum (silver staining). Molecular mass markers (in kDa) are shown on the left-hand side. Input is an initial cytosolic fraction. ft is a flow-through protein fraction for SS and C resins respectively. Arrows show Trx1 bands. CU, Cys–Sec; CC, Cys–Cys; SC, Ser–Cys; SS, Ser–Ser; C, Cys-truncated mutant.

Anton A. Turanov, et al. Biochem J. 2010 September 1;430(2):285-293.
8.
Figure 4

Figure 4. Characterization of TR3-knockdown cells. From: Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

NIH 3T3 cells were stably transfected with pSec hygro (vector) or the vector encoding two siTR3 constructs, siTR3 1.1 and siTR3 1.3. (A) TR3 and TR1 expression in various siTR3 clones and in rat liver cytosolic and mitochondrial fractions assayed by Western blotting with TR1 and TR3 antibodies. (B) Growth rate of TR3-knockdown cells. Growth rates of TR3-knockdown (siTR3 1.1) and control (vector) cells were determined and the results are represented as means ± S.D. of four replicates. (C) UV treatment of TR3-knockdown cells. TR3-knockdown and control (stably transfected with pSEC hygro) cells, assayed 6 or 12 h after exposure to UV irradiation as described in the Experimental section. * Indicates statistical differences compared with the vector control by using the two-tailed Student’s t test (P < 1 × 10−4 for 6 h and P < 0.003 for 12 h respectively).

Anton A. Turanov, et al. Biochem J. 2010 September 1;430(2):285-293.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk