Glutathione peroxidase (GPx) enzymes are generally selenium-containing tetrameric glycoproteins that help prevent lipid peroxidation of cell membranes. GPx enzymes reduce lipid hydroperoxides to alcohols, and reduce free hydrogen peroxide to water. GPx members are among the few proteins known in higher vertebrates to contain selenocysteine, which occurs at the active site of glutathione peroxidase and is coded by the nonsense (stop) codon TGA. There are eight GPx homologs (GPx-1-8). GPx-1, Gpx-2 and Gpx-3 exist as homotetramers. Gpx-4 has a high tendancy to form high molecular weight oligomers. GPx-1 plays an important role in the antioxidant defense of the vascular wall and neural cells in response to oxidative stress. GPx-2 is the major isoform in the lungs and its basal or inducible expression is dependent on Nrf2. GPx-3 is under regulation by hypoxic stress and the expression and deficiency of GPx-3 is associated with cardiovascular disease and stroke. GPx-5 is selenium-independent; it is bound to the acrosome of sperm, where it may protect sperm from premature acrosome reaction in the epididymis.
Background References
1. Maremanda KP et al. Role of Zinc Supplementation in Testicular and Epididymal Damages in Diabetic Rat: Involvement of Nrf2, SOD1, and GPX5. Biol Trace Elem Res 173(2):452-64 (2016).
2. Vilagran I et al. Triosephosphate isomerase (TPI) and epididymal secretory glutathione peroxidase (GPX5) are markers for boar sperm quality.Anim Reprod Sci 165:22-30 (2016).
Western blot analysis of GPX5 on different lysates with Mouse anti-GPX5 antibody (M1701-8) at 1/5,000 dilution.
Lane 1: 293T cell lysate (20 µg/Lane) Lane 2: Mouse testis tissue lysate (40 µg/Lane) Lane 3: Rat testis tissue lysate (40 µg/Lane)
Predicted band size: 25 kDa Observed band size: 20 kDa
Exposure time: Lane 1: 3 minutes; Lane 2-3: 10 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (M1701-8) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunocytochemistry analysis of U-937 cells labeling GPX5 with Mouse anti-GPX5 antibody (M1701-8) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-GPX5 antibody (M1701-8) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.
Flow cytometric analysis of U-937 cells labeling GPX5.
Cells were fixed and permeabilized. Then stained with the primary antibody (M1701-8, 1/1,000) (red) compared with Mouse IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Mouse IgG Secondary antibody (HA1125) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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