Enhanced chemiluminescence assay

Using a Chrono-Log aggregometer, after incubation with vehicle (0.4% DMSO) or 50 μM of Col003 for 5 minutes at 37°C, washed platelets (3 × 10⁸ cells/mL) were stimulated by collagen (2 μg/ml) for 3 minutes. Aggregated platelets were lysed by RIPA lysis buffer containing protease and phosphatase inhibitor cocktail. Protein samples were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride membranes. After incubation with primary antibodies and the corresponding secondary antibodies, the samples were then visualized by enhanced chemiluminescence assay (Beyotime Biotechnology, Shanghai, China). Membranes were stripped and re-probed for β-actin as loading control.

Total protein was collected from tissues or cells with RIPA buffer (Thermo Fisher Scientific). Nuclear and cytoplasmic proteins were isolated from cardiac tissues or H9c2 cells using a NE-PER^™ reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Equal amounts of total protein (approximately 30 μg) were separated using 10% SDS-PAGE and transferred to PVDF membranes (Merck, MA, USA). After blocking with 5% no-fat milk, membranes were incubated with antibodies against Nrf2 (1:1000, ab92946, Abcam), HO-1 (1:4000, ab68477), Gpx4 (1:3500, ab125066), Acsl4 (1:10000, ab155282), Ptgs2 (1:3000, ab179800), β-actin (1:4000, ab8226), and Lamin B1 (1:800, ab229025) overnight at 4°C. Then membranes were incubated with HRP-labeled secondary antibodies after washing three times in TBST. Immunoblot was observed using an enhanced chemiluminescence assay (Beyotime).