Extended Data Fig. 3: Identification of LYN as a succination target for fumarate.
From: Fumarate suppresses B-cell activation and function through direct inactivation of LYN
a, The schematic representation of proteome labelling, enrichment, and digestions to provide probe-labelled peptides for MS analysis. LC-MS/MS, liquid-chromatography-MS/MS. b, Gene Ontology (GO) terms enrichment of functional pathways by competitive activity-based protein profiling (P < 0.05). c, B cell lysates were labeled with IA-alkyne probe without (-) or with DMF (100 µM) competition. Then the enriched proteins as well as input proteins were immunoblotted with LYN antibody. d, Related to Fig. 3d. Purified LYN proteins was treated with DMSO (Ctrl) or increasing concentrations of DMF for 30 minutes and LYN kinase activity was measured by ELISA. e and f, Recombinant LYN with DMSO (up left panel), FA (1 mM), MMF (200 µM), or DMF (100 µM) treatment for 30 minutes was digested with trypsin and analyzed by LC-MS/MS for the position(s) of succination (f). Sequences for all the six detected peptide species covering the succinated residues are shown (e). The MASCOT score obtained for each peptide is listed accordingly. g, Purified wild-type (WT) LYN and LYN-C381S proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining (left panel). The proteins were incubated with DMSO (-) or DMF (100 µM) for 30 minutes to assay LYN kinase activity by ELISA (right and bottom panels). The data in d and g are normally distributed, and the differences were evaluated by using 2-tailed Student’s t test. Data are the mean ± SD. Each experiment was performed three independent times. *P < 0.05, **P < 0.01, ***P < 0.001, based on Student’s t-test.
