Fig. 3
Impact of NAA10 H16P-V5 on NatA complex formation, enzymatic activity and cellular stability. a Western blot analysis of NAA10 WT-V5 and NAA10 H16P-V5 subjected to V5-immunoprecipitation from HeLa cells. V5- and NAA15 antibodies were used for protein detection and NAA10-V5 and NAA15 protein bands were quantified. b Nt-acetylation assay showing catalytic activity of NAA10 H16P-V5 and NAA10 WT-V5. The catalytic activity towards the NatA substrate SESS24 and monomeric NAA10 substrate EEEI24 was normalized to the amount of NAA15 and NAA10-V5, respectively. Negative controls contained either β-gal-V5 or no peptide. The Nt-acetylation assay shown is representative of three independent setups, each with three technical replicates. c Western blot analysis of cell lysates from 0 h (no treatment) and 2, 4 and 6 h after protein synthesis was inhibited by cycloheximide treatment. V5-tag antibody was used to detect NAA10-V5 protein and β-tubulin antibody was used for loading control. d Stability curve displaying the relative amount of NAA10-V5 present at each time point after cycloheximide treatment. The band intensities were quantified from the Western blot (C) and the band intensities of NAA10-V5 variants at each time point were normalized to both the loading control and time point 0 h of the respective variant. The stability curve is representative of three independent setups