Fig. 1: PHF2 is palmitoylated at cysteine 23.

a Levels of palmitoyl-coenzyme A (palmitoyl-CoA) were quantified using liquid chromatography-mass spectrometry (LC-MS) in HepG2 cells treated by palmitic acid (PA) treatment for 24 h. Mean ± SD (n = 3 independent samples); *P < 0.05. Statistical analyses were based on a two-tailed unpaired t test. The exact p-values are presented in Supplementary Data 2. b Hepatocellular carcinoma (HCC) cells were pre-treated with 2-bromopalmitate (2-BP, 50 µM) or dimethyl sulfoxide (DMSO) for 24 h and incubated with PA for 24 h, followed by MG132 (10 µM) for 8 h. Immunoprecipitation was performed using an anti-PHF2 antibody. The precipitated proteins were subjected to an acyl-biotin exchange (ABE) assay using hydroxylamine (HAM) treatment to remove PA from palmitoylated cysteine residues. Free cysteines were labeled with BMCC-Biotin. Finally, palmitoylated proteins were detected using streptavidin-HRP. n = 3 independent experiments. IP: immunoprecipitation; Palm-PHF2: palmitoylated PHF2. c The palmitoylation site within PHF2 was identified using LC-MS analysis. The y and b fragments detected are as indicated in the sequence. The palmitoylation of cysteine 23 residue (C23) in PHF2 was identified based on the shift of peptide peaks. d After transfection with wild-type (WT) PHF2 or C23A-mutated PHF2 plasmids, HCC cells lysates were subjected to western blotting. n = 3 independent experiments. e HCC cells were transfected with Flag-PHF2-WT or Flag-PHF2-C23A mutant. The expressed proteins were immunoprecipitated with Flag affinity beads and subjected to ABE assay. The palmitoylated proteins were detected using western blotting. n = 3 independent experiments. Source data are provided as a Source Data file.