Western Blot Analysis of Autophagy and Ferroptosis Markers

Cultured cells were harvested and lysed in RIPA buffer containing 1% protease inhibitor. Western blot assays were performed as previously described^{44 (link)}. The following antibodies were used: anti-BRD4 (ab75898, 1:1000) and anti-GPX4 (ab125066, 1:1000) were purchased from Abcam (Cambridge, MA, USA); anti-G9a (#3306, 1:1000), anti-SIRT1 (#9475, 1:1000), anti-LAMP1 (#9091, 1:1000), anti-S6 kinase (#2708, 1:1000), anti-LC3B (#3868, 1:1000), anti-SQSTM1/p62 (#8205, 1:1000), anti-ATG5 (#12994, 1:1000), anti-ATG7 (#8558, 1:1000), and anti-FTH1 (#4393, 1:1000) were purchased from Cell Signaling Technology; and anti-β-tubulin, used as the internal control, was purchased from Santa Cruz Biotechnology (TX, USA, CAS: KM9002T, 1:1000). After incubation with primary antibodies, membranes were incubated with goat anti-mouse IgG H&L (horseradish peroxidase (HRP)-conjugated) or goat anti-rabbit IgG H&L (HRP-conjugated) for 1.5 h at room temperature. Immunoreactions were then detected with a FluorChem HD2 imager (Protein Simple, USA).