Immunoblotting Analysis of Cell Signaling Proteins

Immunoblotting was performed as described previously^{49 (link),50 (link)}. Briefly, cell pellets were lysed using RIPA lysis buffer (Millipore, #20-188), and the protein concentration was determined by a Bradford assay (Bio-Rad, #500-0006) using a NanoDrop 2000 (Thermo Fisher Scientific). For immunoblot analysis, 15-30 μg of protein was used along with antibodies against phospho-Rb ([S780] Cell Signaling Technology, #9307, 1:1,000), RB1 (Cell Signaling Technology, #9309, 1:1,000), CDK1 (Cell Signaling Technology, #9116, 1:1,000), ACSL4 (Santa Cruz Biotechnology, #sc-271800, 1:1,000), GPX4 (R&D Systems, #MAB5457, 1:1,000), SLC7A11 (Cell Signaling Technology, #12691, 1:1,000), FSP1 (Santa Cruz Biotechnology, #sc-377120, 1:300), DHODH (Proteintech, #14877-1-AP, 1:1,000), DGAT1 (Santa Cruz Biotechnology, #sc-271934, 1:300), vinculin (Sigma, #V4505, 1:50,000), and tubulin (Sigma, #T9026, 1:10,000), Goat anti-rabbit IgG secondary antibody (Thermo Scientific, #31460, 1:5,000), Goat anti-mouse IgG secondary antibody (Proteintech, #SA00001-1, 1:5,000). The uncropped scans of the immunoblots used in this study are shown in the Source Data file.