Fig. 2: GPX4 is palmitoylated at Cys 66.

a Schematic of the workflow for identifying palmitoylated proteins using the click chemistry assay and LC-MS/MS. b Mass spectrometry results from three independent samples were used to identify palmitoylated proteins. The numbers in the figure represent the palmitoylated proteins identified in each sample (with proteins identified in the HAM group excluded). c Peptide spectral counts for GPX4 in control and 2-BP treated groups, with and without HAM. Palmitoylation analysis of Flag-GPX4 in A375 (d) and 293T (e) cells using the ABE assay, with and without HAM. f–h Palmitoylation of Flag-GPX4 in A375 cells pre-treated with 2-BP and incubated with Alk14, analyzed using the click chemistry assay with and without HAM (f). Quantitative analysis of GPX4 palmitoylation (g) and total GPX4 levels (h). i, j Palmitoylation analysis of exogenous GPX4 and its mutants in A375 cells using the ABE assay with and without HAM (i). Quantitative analysis of GPX4 palmitoylation (j). Immunoblot detection (k) and quantification (l) of GPX4 in A375 cells infected with Flag-GPX4-WT or mutant constructs. m Cell viability analysis in A375-GPX4-KO cells infected with WT or mutant Flag-GPX4 constructs, pre-treated with 2-BP, and then treated with RSL3. Data are presented as mean ± SD. Statistical analysis: two-way ANOVA with Tukey’s test for (c, j); one-way ANOVA with Tukey’s test for (g, h, l). Sample sizes: (c, g, h, j, l, m) n = 3 independent experiments. Image in d, e, f, i, k shows representative results from three independent experiments.