Thioredoxin reductase 1 trxr1

Antioxidant protein levels in PBMCs were determined by Western blot analysis. Twenty-microgram protein aliquots were loaded in each lane of an sodium dodecyl sulfate (SDS) polyacrylamide gel (15% acrylamide) and electrophoresed by molecular weight at 200 V for 90 min. Bands were electrotransferred onto a nitrocellulose membrane by using Trans-Blot^® Turbo™ Transfer System (Bio-Rad, Segrate, Milan, Italy). The membrane was blocked (5% non-fat powdered milk in PBS, pH 7.5, containing 0.1% Tween 20) for 5 h and incubated with the corresponding primary monoclonal antibody. Antibodies anti-catalase (CAT) (1:1000, rabbit), Mn superoxide dismutase (MnSOD) (1:1000, mouse), glutathione reductase (GRd) (1:1000, mouse), glutathione peroxidase (GPx) (1:200, mouse), thioredoxin reductase 1 (TrxR1) (1:200, goat) and uncoupling protein 3 (UCP3) (1:500, mouse) were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Blots were then incubated with a secondary peroxidase-conjugated antibody (1:10,000) against specific primary antibody. Development of immunoblots was performed using an enhanced chemiluminescence kit (Immun-Star^® Western C^® Kit reagent, Bio-Rad Laboratories, Hercules, CA, USA). Protein bands were visualized using the image analysis program Quantity One (Bio-Rad). Precision Plus Protein Kaleidoscope™ (Bio-Rad) was used as a molecular weight marker.

Total protein was extracted from three different ejaculates per boar (300 × 10⁶ spermatozoa) using the complete RIPA lysis buffer. After protein quantification, equal amounts of extracted proteins per each boar were pooled according to the ejaculate (n = 3 pools) and total of 30 μg/pool was loaded onto wells of SDS-PAGE (4-12.5% NuPAGE) gels for resolution at room temperature. In-gel proteins were transferred onto PVDF membranes and the immunoblotting procedure was performed according to the anti-rabbit WesternBreeze™ Chromogenic Detection kits (Thermo Fisher Scientific Inc.). Membranes were incubated 60 min with selected primary antibodies (1/500 dilution).
In both immunological techniques, primary antibodies raised against human aquaporin 1 (AQP-1) and 7 (AQP-7), sodium/glucose co-transporter 5 (GLUT-5), plasminogen (PLG), protamine-1 (PRM-1), and thioredoxin reductase 1 (TRXR-1) purchased at Santa Cruz Biotechnology, Inc. were used. Otherwise indicated, all procedures took place at room temperature.