Fig 1: The N-terminus of CFL1 but not internal lysine is conjugated by SUMO1.a Diagram illustrating CFL1 SUMOylation identification using IP-MS. Endogenous CFL1 proteins were purified from Neuro-2a cells expressing His-SUMO1E93R through De-IP with anti-CFL1 antibody. IP products underwent trypsin digestion and LC-MS/MS analysis. b MS/MS spectrum of a tryptic peptide containing glutamine-threonine-glycine-glycine (QTGG) preceding the N-terminal methionine of CFL1, determined by collision-activated dissociation (CAD). c Internal lysines not required for CFL1 SUMOylation. Lysates from CHO-K1 cells, transfected with CFL125KR-HA, His-SUMO1, and Myc-Ubc9, were subjected to De-IP and IB. d (left) N-terminal acetylation inhibits SUMOylation. Lysates from CHO-K1 cells transfected with CFL1WT-HA, His-SUMO1, and Myc-Naa60, were subjected to De-IP and IB. (right) Quantification of Western blots. Mean ± SEM; ***p < 0.001, by two-tailed t test. e Diagram of two methylated recombinant CFL1 proteins: MeM1+25K-CFL1 (top) and Me25K-CFL1 (bottom). f (left) N-terminal α-NH2 group of CFL1 modified by SUMO1 in vitro. Purified CFL1 and methylated variants were incubated with E1, E2, SUMO1, and ATP in various combinations at 37 °C for 1 h, analyzed by IB and CBB staining. (right) Quantification of CBB gel. Mean ± SEM; ****p < 0.0001, by one-way ANOVA with Tukey’s multiple comparison test. g Internal CFL1 lysines were not SUMOylated in vitro. Purified CFL1 and GST-CFL1 underwent in vitro SUMOylation, followed by thrombin digestion at room temperature for 12 h (see Supplementary Fig. 5d). All blots represent ≥3 independent experiments. Exact p values and source data provided in Source Data file.