Fig. 3

Effect of ROS on protein synthesis in neurons. a Schematic drawing of the SUnSET method. Puromycin labels newly synthesized proteins. Puromycin is detected by anti-puromycin antibody. b SUnSET assay in WT and SMA MNs (N = 5). ACTB was used as loading control. c Schematic drawing of the Click-iT AHA method. L-azidohomoalanine (L-AHA) is incorporated into newly synthesized proteins, followed by click chemistry reaction with biotin alkyne. Biotin-labeled proteins can be detected by western blot analysis. d Click-iT AHA assay in WT and SMA MNs (N = 4). ACTB was used as loading control. e Scheme of experimental design. 10DIV MNs were treated with 10 µM NAC or 50 mM pyruvate for 1 h. Cells were incubated with 1 μM puromycin for 30 min before analysis. f, g SUnSET assay after ROS modification in (f) WT MNs (N = 5) and (g) SMA MNs (N = 5). Blue circles represent data from WT and red squares represent SMA MNs. Two-tailed unpaired t-tests with Holm-Bonferroni correction for multiple comparisons were used to determine statistical significance. All bar graphs depict the mean ± S.D., n.s. p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001