Figure 7.

Ectopic expression of SIRT4 impacts negatively on acetylated α-tubulin (K40) during mitosis. HEK293 cells stably expressing eGFP, SIRT4-eGFP, or mutants thereof were either asynchronously grown (a) or subjected to Ro3306-mediated G₂ synchronization (b) followed by immunoblot analysis of HDAC6, acetylated α-tubulin (K40), and α-tubulin. Expression of eGFP, SIRT4-eGFP, or mutants thereof was analyzed in a second immunoblot (lower panels in a and b). Probing against GAPDH was employed as loading control. Relative HDAC6 amounts and acetylated α-tubulin (K40)/α-tubulin levels were quantified by densitometric analysis (c). To evaluate statistical significance one-way ANOVA followed-up by Tukey’s test was performed (three independent experiments; * p < 0.05; ** p < 0.01). All P-values of the analysis of acetylated α-tubulin (K40)/α-tubulin levels (+Ro3306) refer to comparison with SIRT4-eGFP.