Rab22a-NeoF1 fusion protein is acetylated at lysine 7 (K7). (A) HEK293T cells transfected with Rab22a-NeoF1-SFB for 48 h, and cell lysates were subjected to immunoprecipitation (IP) with anti-Flag agarose, the IP complex was then analyzed by mass spectrometry. MS spectra of Ac-K containing the 5-ELKVCLLGDTGVGK-18 peptide obtained after trypsin digestion of the IP complex. (B, C) HEK293T cells transiently transfected with Rab22a-NeoF1-SFB or its K7R mutant for 24 h were treated with TSA (5 µM), NAM (5 mM), or both for 8 h. Cell lysates were subjected to IP with anti-Flag agarose, and then were analyzed by Western blotting. (D) ZOS-M cells were treated with both TSA (5 µM) and NAM (5 mM) for 24 h, cell lysates were subjected to IP using anti-K7ac-Rab22a-NeoF1 antibody, and then were analyzed by Western blotting using mAb RAD5-8.