Figure 5

Introducing a lysine ubiquitin acceptor site does not affect p16 stabilization by Nt-acetylation. (A) HEK 293T cells were transfected to express indicated p16-HA variants in WT or + K versions 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. (B, C) 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 B shows a representative Western blot image, panel C shows the quantification. Note that the p16-HA (+ K) variants were studied in side-by side experiments with the p16 variants described in Fig. 4A, B. The Western blot and quantification for the ~ GY variant depicted in Fig. 4A, B are shown here again for comparison purposes (n = 3; mean ± SEM). (D) Based on the data in panel C, the half-lives of the indicated p16-HA variants were determined (n = 3; mean ± SEM; Ratio paired t-test, two-tailed).