Sc 376858

Protein extracts were separated by 1D gel electrophoresis using NuPAGE 4–12% (w/v) Bis-Tris midi gels (Invitrogen, USA), and transferred onto PVDF membranes using an iBlot 2 transfer system (Invitrogen, USA), according to the manufacturer’s specifications. Immunoblots were performed to detect FLAG tag (1:1000, D6W5B, Cell Signaling Technology, USA), LRP8/APOER2 (1:2000; ab108208, Abcam, UK), β-ACTIN (1:5000, AC-15, Sigma-Aldrich, Australia), GPX4 (1:1000, ab125066, Abcam, USA), Notch1 (1:1000, D1E11, Cell Signaling Technology, USA), PS1 (1:1000, D39D1, Cell Signaling Technology, USA), or PS2 (1:1000, D30G3, Cell Signaling Technology, USA), SELENOP (B-9, Mouse mAb; sc-376858, Santa Cruz Biotechnology, Inc., USA), cleaved caspase 3 (Asp175; 1:1000, 5A1E, Rabbit mAb 9664; Cell Signaling Technology, USA), or cleaved caspase 7 (Asp198; 1:1000, D6H1, Rabbit mAb 8438) followed by appropriate HRP-conjugated secondary antibodies (1:5000, Thermo, Australia). Chemiluminescence was detected using Pierce ECL (Thermo, Australia) and visualized using the LAS-3000 Imaging System (Fujifilm, Japan) or Odyssey® Fc Imaging System (LI-COR Biosciences, Lincoln, NE). Representative blots for all proteins (including β-actin) are shown. Original western blots for all relevant figures are shown in “Supplementary Material—Original Blots”.

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).

The skeletal muscle tissues were homogenized with the cell disruption buffer (RIPA lysis Buffer, Beyotime, Shanghai, China), then the total protein concentration was measured using the BCA kit (Jiancheng Bioengineering, Nanjing, China). The subsequent Western blot process was performed as previously described [11 (link),29 (link)]. The primary antibodies were used at the following dilutions: HSP70 (1:5000; ab5439; Abcam, Cambridge, UK), GPX1 (1:1000; 616958; Zen BioScience, Chengdu, China), GPX3 (1:2000; sc-58361, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPX4 (1:2000; 513309, Zen BioScience, Chengdu, China), SELENOP (1:2000; sc-376858, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SELENOS (1:1000, 15591-1-AP, ProteinTech Group, Chicago, IL, USA) and GAPDH (1:5000; 200306-7E4, Zen BioScience, Chengdu, China).