Fig. 5. Validation of NatA-catalysed acetylation of Nt-Cys JunB.
a JUNB-FLAG was overexpressed in HAP1 ADO KO cells and treated with either siCTRL, siNAA10 or siNAA20. Cells treated with siCTRL as well as empty vector (EV) were included as controls. Anti-NAA10 and anti-NAA20 were used to verify successful knockdown and disrupted NatA/NatB complexes, whereas anti-JUNB was used to verify successful overexpression and IP. Protein gel image included as loading control. #1 and #2 represent two independent samples. b The number of peptide-spectrum matches (PSMs) identified by mass spectrometry for JUNB N-terminal peptides as well as two selected JUNB internal peptides. JUNB peptides designated XXXXn, where XXX are the first four amino acids of the peptide and n is the length of the peptide fragment. Data represent 2 independent biological samples; black dots represent each independent replicate. c Example MS/MS spectra for the N-terminal peptide of JUNB in its Nt-acetylated state from siCTRL treated cells and unacetylated state from siNAA10 treated cells showing bx (blue) and yx (purple) +1 ions with their respective mass/charge ratio and intensity. Free thiols in protein samples were alkylated prior to digestion and LC/MSMS, so Nt‑Cys bears a carbamidomethyl group adding 57 Da to the expected mass of b fragment ions. For expanded view see Supplementary Fig. 6. d S. cerevisiae strains WT, naa10∆ and naa20∆, with the last two lacking functional NatA and NatB, respectively, were transformed with a JUNB-FLAG plasmid or control plasmid. Protein gel image included as loading control. e The number of PSMs identified by mass spectrometry for JUNB N-terminal peptides as well as two selected JUNB internal peptides. JUNB peptides designated XXXXn, where XXXX are the first four amino acids of the peptide and n is the length of the peptide fragment. Data represent 3 independent biological samples ± standard deviation; black dots represent each independent replicate. MW units = kDa. Source data are provided as a Source Data file.