Extended Data Fig. 2: Symmetric dimethylation of GPX4 at R152.
From: PRMT5-mediated arginine methylation stabilizes GPX4 to suppress ferroptosis in cancer
a, GPX4 immunoprecipitated from HEK-293T were separated by SDS–PAGE, and collected for mass spectrometry analysis. Mass spectrometry analysis shows the arginine methylation of R152 in GPX4. b-d, Titration of the indicated GPX4 peptides with or without R152 systematic dimethylation (b) demonstrates that the generated GPX4 R152-me2s antibodies specifically recognize the GPX4-R152-SDMe epitope in the dot blot analysis (c), and IB analysis of anti-Flag IPs derived from HEK-293T cells transfected with wild-type or R152K mutant Flag-GPX4 (d). e-f, Schematic representation of the genome sequence to generate GPX4-R152K CRISPR knock-in cells (e, upper). Identification of the potential knock-in mutants. Genomic DNA containing GPX4-R152K mutation was amplified by PCR and digested with BamHI (e, lower). Confirmation of the correct mutation of GPX4-R152K by Sanger DNA sequencing (f). g, IB analysis of the WCL derived from indicated HeLa after treated with MG132 (20 μM) for 12 h. h-j, IB analysis (h, left) of Parental and homozygous GPX4-R152K mutant HeLa cells, IB analysis (h, right), CHX chase assay (i), in vitro ubiquitination assay (j) of HeLa GPX4-knockout cells transfected with wild-type or the R152K mutant Flag-GPX4. Where indicated, MG132 (10 μM, 12 h) and CQ (50 nM, 24 h) were add in medium. All of the experiments, from c to j, were repeated at least twice independently, yielding identical outcomes.
