Western Blot Analysis of Protein Expression

Total protein was extracted from RAW264.7 cells or alveolar macrophages according to published methods [60 (link)]. The membranes were subjected to an overnight incubation process at a controlled temperature of 4 °C, during which they were treated with primary antibodies. These antibodies were carefully diluted to a ratio of 1:1000 and included NCOA4 (Abclonal), GPX4 (Abclonal), FTH1 (ABclonal), LC3 (ABclonal) or β-Actin (ABclonal). The membranes were then incubated with secondary antibodies (diluted 1:1000, ABclonal, China) at room temperature for 1 h. Signals were detected using an advanced enhanced chemiluminescence system manufactured by Thermo Scientific, and relative protein expression was finally quantified with ImageJ software.