*4.7. Western Blot Analysis*

Plasma samples were processed as such, while the isolated lymphocyte pellet was homogenized and centrifuged at 10,000× *g* for 10 min. The supernatant was then used for analysis after determination of protein content. Proteins extracted for each sample, at equal concentration (50 μg), were boiled for 3 min in sample buffer (containing 40 mM Tris-HCl pH 7.4, 2.5% SDS, 5% 2-mercaptoethanol, 5% glycerol, 0.025 mg/mL of bromophenol blue) and then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred onto nitrocellulose membrane (BIO-RAD Hercules, CA, USA) in transfer buffer containing 0.05% of SDS, 25 mM di Tris, 192 mM

glycine and 20% v/v methanol. The transfer of the proteins on the nitrocellulose membrane was confirmed by staining with Ponceau Red which was then removed by 3 washes in PBS (phosphate buffered saline) for 5 min/each. Membranes were then incubated for 1 h at room temperature in 20 mM Tris pH 7.4, 150 mM NaCl and Tween 20 (TBS-T) containing 2% milk powder and incubated with appropriate primary antibodies, namely anti-γ-GC liase anti-Hsp70, anti-HO-1, anti-Sirt-1, anti-Trx and anti-HNE polyclonal antibody (Santa Cruz Biotech. Inc.), overnight at 4 ◦C in TBS-T. The same membrane was incubated with a goat polyclonal antibody anti-beta-actin (SC 1615 Santa Cruz Biotech. Inc., CA, USA, dilution 1:1000) to verify that the concentration of protein loaded in the gel was the same in each sample. The excess of unbound antibodies was removed by 3 washes with TBS-T for 5 min. After incubation with primary antibody, the membranes were washed 3 times for 5 min in TBS-T and then incubated for 1 h at room temperature with the secondary polyclonal antibody conjugated with horseradish peroxidase (dilution 1:500). The membranes were then washed 3 times with TBS-T for 5 min. Finally, the membranes were incubated for 3 min with SuperSignal chemiluminescence detection system kit (Cod 34080 Pierce Chemical Co, Rockford, USA) to display the specific protein bands for each antibody. The immunoreactive bands were quantified by capturing the luminescence signal emitted from the membranes with the Gel Logic 2200 PRO (Bioscience) and analyzed with Molecular Imaging software for the complete analysis of regions of interest for measuring expression ratios. The molecular weight of proteins analyzed was determined using a standard curve prepared with protein molecular weight.

#### *4.8. Glutathione and Glutathione Disulfide Assay*

GSH and GSSG were measured by the NADPH-dependent GSSG reductase method as previously reported in Calabrese et al. 2010. Lymphocytes were homogenized on ice for 10 s in 100 mM potassium phosphate, pH 7.5, which contained 12 mM disodium EDTA. For total glutathione, an aliquots (0.1 mL) of homogenates were immediately added to 0.1 mL of a cold solution containing 10 mM DTNB and 5 mM EDTA in 100 mM potassium phosphate, pH 7.5. The samples were then mixed by tilting and centrifuged at 12,000× *g* for 2 min at 4 ◦C. An aliquot (50 μL) of the supernatant was added to a cuvette containing 0.5 U of GSSG reductase in 100 mM potassium phosphate and 5 mM EDTA, pH 7.5 (buffer 1). After 1 min of equilibration, the reaction was initiated with 220 nmol of NADPH in buffer 1 for a final reaction volume of 1 mL. The formation of a GSH-DTNB conjugate was then measured at 412 nm. The reference cuvette contained equal concentrations of DTNB, NADPH, and enzyme, but not sample. For assay of GSSG, aliquots (0.5 mL) of homogenate were immediately added to 0.5 mL of a solution containing 10 mM N-ethylmaleimide (NEM) and 5 mM EDTA in 100 mM potassium phosphate, pH 7.5. The sample was mixed by tilting and centrifuged at 12,000× *g* for 2 min at 4 ◦C. An aliquot (500 μL) of the supernatant was passed at one drop/s through a SEP-PAK C18 Column (Waters, Framingham, MA) that had been washed with methanol followed by water. The column was then washed with 1 mL of buffer 1. Aliquots (865 μL) of the combined eluates were added to a cuvette with 250 nmol of DTNB and 0.5 U of GSSG reductase. The assay then proceeded as in the measurement of total GSH. GSH and GSSG standards in the ranges between 0 to 10 nmol and 0.010 to 10 nmol, respectively, added to control samples were used to obtain the relative standard curves, and the results were expressed in nmol of GSH or GSSG, respectively, per mL or mg protein.

## *4.9. Spontaneous Ultraweak Chemiluminescence Assay*

Measurement of chemiluminescence in blood samples was accomplished according to the method of Flecha et al. 1991 [59]. Briefly, aliquots (0.5 mL) of plasma were diluted 1:1 with 30 mM phosphate buffer (pH 7.4), whereas lymphocyte pellet was homogenized and centrifuged at 10,000× *g* for 10 min. Before aliquots (0.5 mL) of the supernatant were taken and diluted 1:1 with 30 mM phosphate buffer (pH 7.4) at 0–4 ◦C and centrifuged at 10,000 *g* for 3 min at 0–4 ◦C. Then spontaneous ultraweak chemiluminescence (UCL) was measured in the supernatant at 30 ◦C with a Turner TD 20/20 luminometer. The sensitivity was adjusted to 50%, and results were expressed as luminescence units/mg protein.
