Prewashed streptavidin beads

HL60 received RSL3 for 6 h to promote ferroptosis. THP-1 cells were pretreated with PMA (40 pmol/10⁶ cells) for 3 days and incubated with ferroptotic cells for 1.5 h at 37 °C. Unengulfed HL60 cells were removed. Then, the THP-1 cells were treated with 50 μM biotin or SAPE-biotin in RPMI 1640 (without FBS) for 4 h, and then harvested and incubated with prewashed streptavidin beads (Thermo Fisher Scientific) overnight at 4 °C on a shaker. Proteins interacted with SAPE-biotin were eluted by Laemmli buffer containing 500 μL 6 M urea, 25 μL 200 mM DTT, and 25 μL 500 mM IAA in dark at room temperature for 30 min. The eluent was incubated with 150 μL 2M urea, 150 μL 1 mM CaCl₂, and 1 μL trypsin (1 μg/μL) at 37 °C overnight. After that, the protein samples were purified by ODS C18 SPE column (Agilent) and analyzed by LC–MS/MS. Samples were then analyzed in a data-dependent acquisition mode by the LC−MS/MS, equipped with an EASY-nLC 1200 (Thermo Fisher Scientific) HPLC system and Orbitrap Fusion Lumos (Thermo Fisher Scientific) mass spectrometer. For LC separation, tryptic peptides were sequentially injected into an Acclaim PepMap 100 C18 column (100 μM × 2 cm, 5 μM, Thermo Fisher Scientific, P/N:164564) and an Acclaim PepMap 100 C18 column (50 μM × 15 cm, 2 μM, Thermo Fisher Scientific, P/N:164943).

HL60 received RSL3 for 6 h to induce ferroptosis. THP-1 cells were pretreated with PMA (40 pmol/10⁶ cells) for 3 days and incubated with ferroptotic cells for 1.5 h at 37 °C. Unengulfed HL60 cells were removed. Then, the THP-1 cells were treated with 50 μM biotin or SAPE-biotin in RPMI 1640 (without FBS) for 4 h, and then harvested and incubated with prewashed streptavidin beads (Thermo Fisher Scientific) overnight at 4 °C on a shaker. The beads were collected and heated to 100 °C for 10 min, and then loaded on SDS-PAGE.
For protein gel staining, gels were incubated with Coomassie Brilliant Blue staining solution for 6 h, and then washing five times with 1% phosphoric acid (H₃PO₄).
For western blotting, proteins were transferred from gel to Immobilon-P PVDF membrane (MilliporeSigma). Proteins expression were detected using TLR2 antibody and visualized using secondary antibody conjugated with HRP and Pierce ECL western blotting substrate (Thermo Fisher Scientific) as the substrate of HRP. The immunoblot was detected by Tanon 5200 Chemiluminescence Image Analysis System (Tanon Science & Technology).