Ab59546

Porcine mammary epithelial cells were seeded into six-well plates at 2 mL/well at 5 × 10⁴ cells/mL and cultured in a complete medium at 37°C and 5% CO₂ for 48 h. Then, the cells were treated with different levels of Se-Met (0, 0.5, 1, 2, or 4 μM) for 48 h. After that, the cells were collected and homogenized in a RIPA lysis buffer (Beyotime, Nanjing, China). Western blot analysis was performed according to the procedures described in our previous study (26 (link)). The primary antibodies were as follows: (1) anti-SEPHS2 antibody (1:1,000, ab153878, Abcam, MA, USA), (2) anti-SELENOP antibody (1:1,000, sc-376858, Santa Cruz, CA, USA), (3) anti-GPX1 antibody (1:1,000, ab59546, Abcam, MA, USA), (4) anti-TXNRD1 antibody (1:1,000, ab78629, Abcam, MA, USA), and (5) anti-β-actin (1:1,000, bs-0061R, Bioss, Beijing, China).

Rat knee joint tissues were homogenized and lysed in a protein extraction reagent (Tissue protein extraction reagent, Pierce, Rockford, US). Western blot analysis was performed as described previously [29 (link)]. The primary antibodies to transferrin (17435-1-AP; Proteintech), catalase (ab209211; Abcam), SOD1 (ab16831; Abcam), SOD2 (ab68155; Abcam), SOD3 (ab21974; Abcam), glutathione peroxidase 1 (ab59546; Abcam), glutathione peroxidase 2 (MAB5470; R&D Systems), transthyretin (ab9015; Abcam), NF-κB (8242S; Cell Signaling), IL-1β (ab9722; Abcam), RANKL (sc-9073; Santa Cruz), and GAPDH (sc-20357; Santa Cruz) were obtained commercially. After extensive washing, appropriate secondary antibodies were used in the experiments, including anti-rabbit IgG antibody conjugated with horseradish peroxidase (7074S; Cell Signaling), anti-mouse IgG antibody conjugated with horseradish peroxidase (7076P2; Cell Signaling), or anti-sheep IgG antibody conjugated with horseradish peroxidase (313-035-003; Jackson ImmunoResearch).

Total liver protein lysates were obtained and separated in 10% sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). Gels were then blotted onto PVDF Amersham Hybond-P membranes (GE Health-care, Buckinghamshire, UK) and incubated with their corresponding antibodies (A2228, A9917, C0979 and S2147 from Sigma–Aldrich, St. Louis, Missouri, USA; Ab59546 from Abcam, Cambridge, UK; sc-2004, sc-2490 and sc-32886 from Santa Cruz Biotechnology, Dallas, Texas, USA). β-actin was used as loading control. Blots were developed by enhanced chemiluminescence using an Amersham ECL Plus Western Blotting Detection Reagent (GE Health-care, Buckinghamshire, UK) according to manufacturer’s instructions.