Journal: Free radical biology & medicine

Article Title: Peroxiredoxin VI Oxidation in Cerebrospinal Fluid Correlates with TBI Outcome

doi: 10.1016/j.freeradbiomed.2014.04.002

Figure Lengend Snippet: Panel A: Peroxidase activity of continuously oxidized with H2O2 (2.5 μM) purified Prdx6 (0.5 μg, see Fig. 3E, with addition of GSH-loaded GSTP1-1) using H2O2 (20 μM) as a substrate in vitro. Panel B: peroxidase activity of purified Prdx6 in vitro and in CSF of control (#10) and TBI A (#20) patients using PLPCOOH or H2O2 (both 20 μM), as a substrate. Prdx6 specific inhibitor MJ33 (~50 μM) was used for peroxidase specificity control. The traces are representative of three independent experiments. Panel C: Effect of TBI-mediated Prdx6 oxidation on neurological deficit of patients at discharge. Data represent mean for: n=8(NDD=1), n=12(NDD=2) and n=4(NDD=3) independent experiments and statistical relevance of differences (ANOVA).

Article Snippet: Non-specific binding was blocked by incubating membranes in TBS-T (Tris-buffered saline with 0.1% Tween-20) containing 5% non-fat dried milk or 5% bovine serum albumin Fraction V. Polyclonal rabbit Prdx6 antibody (1:4,000 dilutions, Strategic Biosolutions, Ramona, CA), anti-GSTP-1-1 (1:500 dilution, MBL, MA) and anti-Prdx6-SO 2(3) H (1:1,000 dilution, Lab Frontier, Japan) were incubated for two hours at room temperature or overnight at 4°C.

Techniques: Activity Assay, Purification, In Vitro, Control

Journal: Free radical biology & medicine

Article Title: Peroxiredoxin VI Oxidation in Cerebrospinal Fluid Correlates with TBI Outcome

doi: 10.1016/j.freeradbiomed.2014.04.002

Figure Lengend Snippet: Prdx6 peroxidase activity in CSF of control (non-trauma) and TBI patients and in vitro using purified E. Coli- expressed Prdx6 and GSH-loaded GSTP1-1 proteins (see Materials and Methods). Data represent mean±SD for 3 independent experiments. Patient number is shown in parenthesis. ND-not detected.

Article Snippet: Non-specific binding was blocked by incubating membranes in TBS-T (Tris-buffered saline with 0.1% Tween-20) containing 5% non-fat dried milk or 5% bovine serum albumin Fraction V. Polyclonal rabbit Prdx6 antibody (1:4,000 dilutions, Strategic Biosolutions, Ramona, CA), anti-GSTP-1-1 (1:500 dilution, MBL, MA) and anti-Prdx6-SO 2(3) H (1:1,000 dilution, Lab Frontier, Japan) were incubated for two hours at room temperature or overnight at 4°C.

Techniques: Activity Assay, Control, In Vitro, Purification

Journal: Free radical biology & medicine

Article Title: Peroxiredoxin VI Oxidation in Cerebrospinal Fluid Correlates with TBI Outcome

doi: 10.1016/j.freeradbiomed.2014.04.002

Figure Lengend Snippet: Panel A: comparison of Prdx6 level in: 1- lung homogenate; 2- TBI CSF; 3 - TBI CSF; 4- control CSF (all lanes contained 20.0 μg of total protein). Panel B: Prdx6 detection in: 1 – plasma (see Materials and Methods) and 2 - RBC lysates from the blood of control patients (SDS PAGE is presented as loading control, 20.0 μg protein per lane). Panel C: dot plot of Hb content in the CSF of control (n=9) and TBI (nTBI A=8, nTBI B=10) patients. Panel D: purified Prdx6 (1.0 μg) monomerization as a result of continuous incubation with 4.0 μM of H2O2 at 37°C in vitro. TCEP reduction of disulfide-based Prdx6 homodimer and quantification (Odyssey Clx, Image Studio 3.1 software) of total reduced monomer normalized for initial (starting material) monomer content before reduction. Panel E: purified Prdx6 (0.5 μg) overoxidation (upper panel, Ab against Prdx6-SO2(3)H) and monomerization (lower panel, same blot with Ab against Prdx6) as a result of its continuous incubation with 2.5 μM of H2O2 at 37°C in vitro. Quantification of Prdx6 overoxidation was fit with sigmoid curve using SigmaPlot 10.0 (SyStat, MA). Quantification of Prdx6 monomeric and dimeric forms was done similar to that in panel D. Data presented as mean±SD for 3 independent experiments. Arrows indicate monomeric (*) and dimeric (**) forms of Prdx6.

Article Snippet: Non-specific binding was blocked by incubating membranes in TBS-T (Tris-buffered saline with 0.1% Tween-20) containing 5% non-fat dried milk or 5% bovine serum albumin Fraction V. Polyclonal rabbit Prdx6 antibody (1:4,000 dilutions, Strategic Biosolutions, Ramona, CA), anti-GSTP-1-1 (1:500 dilution, MBL, MA) and anti-Prdx6-SO 2(3) H (1:1,000 dilution, Lab Frontier, Japan) were incubated for two hours at room temperature or overnight at 4°C.

Techniques: Comparison, Control, Clinical Proteomics, SDS Page, Purification, Incubation, In Vitro, Software

Journal: Free radical biology & medicine

Article Title: Peroxiredoxin VI Oxidation in Cerebrospinal Fluid Correlates with TBI Outcome

doi: 10.1016/j.freeradbiomed.2014.04.002

Figure Lengend Snippet: Panel A and B: Prdx6 detection in CSF of control and TBI patients, respectively (matching SDS PAGE images are presented as a loading control). Panel C: effects of TCEP (~1.0 mM) reduction on Prdx6 monomerization in CSF of TBI patient (#20). Panel D: Prdx6 dimer and GSTP1-1 monomer detection in CSF of control patient #10. Panel E: Prdx6 monomerization/oxidation(red color) and GSTP1-1 dimerization dynamics detection in CSF of TBI patients with mild (#19) and severe (#7) outcome at the time of ER admission and 24 hours later. Prdx6 blots for patients #7 and #19 were stripped and re-probed anti Prdx6-SO2(3)H Ab and detected with red fluorescent secondary Ab. Panel F: Prdx6 detection in collecting duct cells (40 μM of total protein/lane). Arrows indicate monomeric (*) and dimeric (**) forms of Prdx6 and GSTP1-1. The blots (~15 μg of total protein/lane) are representative of three independent experiments. NDD: Neurological Deficits at Discharge.

Article Snippet: Non-specific binding was blocked by incubating membranes in TBS-T (Tris-buffered saline with 0.1% Tween-20) containing 5% non-fat dried milk or 5% bovine serum albumin Fraction V. Polyclonal rabbit Prdx6 antibody (1:4,000 dilutions, Strategic Biosolutions, Ramona, CA), anti-GSTP-1-1 (1:500 dilution, MBL, MA) and anti-Prdx6-SO 2(3) H (1:1,000 dilution, Lab Frontier, Japan) were incubated for two hours at room temperature or overnight at 4°C.

Techniques: Control, SDS Page