Figure 4

Nt-acetylation correlates with stability of the naturally lysineless protein p16. (A, B) HEK 293T cells were transfected to express GFP as a reference protein, together with p16-HA variants carrying indicated N-terminal dipeptide sequences (”Ac-” = Nt-acetylated, “ ~ ” = Nt-acetyl-free). Cells were cultured in presence of CHX (50 μg/ml) for the indicated time periods, followed by immunoblot analysis with antibodies to the HA tag and to GFP. P16-HA signals were quantified and corrected for GFP signal intensity, and the resulting value at the 0 h time point was set to 100%. Data points were connected by a one-phase decay curve fit. Panel A shows a representative Western blot image, panel B shows the quantification (n = 3; mean ± SEM). (C) Based on the data in panel B, the half-lives of the indicated p16-HA variants were determined (n = 3; mean ± SEM, Ratio paired t-test, two-tailed). (D) HEK 293T cells were transfected to express indicated p16-HA variants and FLAG-tagged ubiquitin, followed by lysis under denaturing conditions. Next, p16-HA was immunoprecipitated and the precipitates were analyzed by immunoblotting. A representative image of three independent biological replicates is shown. (E) U-2 OS cells were transfected to express indicated p16-HA variants, followed by DNA staining with propidium iodide, and cell cycle analysis by flow cytometry (n = 3; mean ± SEM, Paired t-test, two-tailed). (F) Cell lysates from cells described in panel E were analyzed by immunoblotting. Spectrin-GFP is a transfection and loading control.