Fig. 2

Activation of macrophages with LPS increases protein malonylation, with GAPDH as a substrate. a Western blot analysis of lysine malonylation (mal-K) in lysates from BMDMs treated with LPS (100 ng/mL) for 24 h. b Most enriched functions associated with LPS-induced malonylated proteins (FDR < 0.05). c Immunoprecipitated GAPDH from untreated and LPS-treated (100 ng/mL, 6, 12 and 24 h) BMDMs and samples probed with an anti-malK antibody (lower panel). GAPDH expression in the immunoprecipitated (upper panel) samples was also examined. d Chemical formula of the MalAMyne probe. e Untreated and LPS-treated (100 ng/mL, 24 h) BMDMs were labelled with MalAMyne (10 µM) or vehicle control. Copper-catalysed click chemistry was performed on the lysates using biotin, followed by immunoprecipitation using streptavidin (strept.). Samples were probed for GAPDH via western blotting. f Purified GAPDH (100 µg/mL) was incubated in the presence of TCEP and malonyl-CoA (or buffer as control) for 1 h at 37 °C, pH 7.5. K-malonylation was assessed by western blotting. g Identification of malonylated sites in immunoprecipitated trypsin-digested GAPDH peptides from untreated and LPS-treated BMDMs (10⁶ cells). Peptides were analysed via LC–MS. h Purified GAPDH was preincubated with TCEP and 25 mM iodoacetamide, 80 mM methyl methanethiosulfonate (MMTS), 5 μM heptelidic acid (HA) or buffer for 30 min at 37 °C, followed by 500 μM malonyl-CoA. K-malonylation was assessed by western blotting. All data shown are representative of three independent experiments