Fig. 5: Itaconation at Cys239 enhances FYN kinase activity.

a Western blotting validation of novel itaconated proteins (FYN, FLT1) identified by PBSP. b Inhibition of ITalk labeling on purified FYN by iodoacetamide (IAA) pre-treatment. Purified FYN (20 μM) was incubated with 10 mM or 20 mM iodoacetamide for 30 min, then treated with 1 mM ITalk for 1 hour. c Tandem MS spectra of peptides containing itaconation potentially on Cys239 of FYN. d ITalk labeling in FYN single-cysteine mutants. e ITalk labeling in a FYN mutant lacking all seven cysteines. In (d, e), HEK293T cells expressing each FYN mutant were treated with 1 mM ITalk for 6 hours. Lysates were subjected to click reaction with azide-biotin, streptavidin enrichment, and anti-MYC blotting. f Domain architecture and structural location of the FYN itaconation site (Cys239, SH2 domain; PDB: 3UF4). g Reduced FYN Tyr531 phosphorylation following 24 hours treatment with 3 mM itaconate or 500 μM 4-OI in Raw264.7 cells. h The impact of itaconate and 4-OI on FYN phosphorylation at Tyr531 in BMDMs. i Time-dependent reduction of FYN Tyr531 phosphorylation in Raw264.7 cells treated with 100 ng/mL LPS. j FYN Tyr531 phosphorylation in WT and IRG1 KO cells following LPS treatment. k Proposed hypothesis on how itaconation of FYN regulates intercellular communications. Created in BioRender. Chu, l. (2025) https://BioRender.com/f0mqqeh. In (a, b, d, e, g, h, i, j), three replicates were performed with similar results. Corresponding uncropped images are shown in the Source Data file.