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. 2017 Jan 19;16(7):649–659. doi: 10.1080/15384101.2017.1281481

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Figure 3. — Immunoassays for YopJ-mediated serine and Lys-acetylation of MARCH8. (a) MS/MS spectrum of MARCH8 synthetic peptide containing Sac71. (b) Specificity of the Sac71 antibody demonstrated by dot blotting. Two acetylated peptides Ac-T(Sac)ITPSSQDICRICHCEGDC-NH2 (Sac71), Ac-KS(ac)PLTEPNFEN KC-NH2 (Sac253), and unmodified peptide Ac-TSITPSSQDICRICHCEGDC-NH2 (S71), Ac-KSPLTEPNFENKC-NH2 (S253) were blotted on NC membrane, detected with the anti-Sac71 polyclonal antibody and stained with IRDye680-conjugated goat anti-rabbit IgG. Anti-Sac71 antibody recognize only Sac71 peptide. (c) Dot-blotting for competition: The acetylated peptide Ac-T(Sac)ITPSSQDICRICHCEGDC-NH2 (Sac71) and non-acetylated peptide Ac-TSITPSSQDICRICHCEGDC-NH2 (S71) were blotted on NC membrane, detected with the anti-Sac71 polyclonal antibody pre-incubated with Sac71 or S71 peptide and visualized with IRDye680-conjugated goat anti-rabbit IgG. Pre-incubation with Sac71 antibody competed away the dot-blot signals while pre-incubation with S71 preserved the signals. (d) Western-blotting for competition. HeLa cell lysates transfected with the truncated MARCH8 (ΔM8) was resolved by SDS-PAGE. Western blots were detected with anti-Sac71 (the left panel) or anti-flag (the right panel) antibody with the competition of the acetylated peptide (Sac71) or the non-acetylated peptide (S71). M: low molecular protein marker (e) Immune assays to prove YopJ-mediated dual acetylation at serine and lysine. HeLa cells were co-transfected with MARCH8 in the presence (+) or absence (−) of YopJ /C172A mutant. M8: transfection with MARCH8 recombinant plasmid. YopJ: cotransfection with both March8 and YopJ recombinant plasmids. C172A: cotransfection with both March8 and YopJ catalytic mutant C172A recombinant plasmids. The MARCH8 was immunoprecipitated with the anti-flag antibody and Western blots were detected with the anti-flag (the left panel), anti-Sac71 (the middle panel) or pan-acetyl - lysine antibodies(the right panel). M: low molecular protein marker (f) YopJ-mediated dual acetylation of MARCH8 was quantified by LI-COR quantitation software. The intensities obtained with anti-Sac71 (Ser-acetylation) and anti-pan-acetyl lysine (Lys-acetylation) antibodies were normalized to that of the anti-flag antibody, data from 3 replicate experiments.