FIG. 2.

There is increased phosphorylation of SIRT2 protein at G₂ and in M phase. SIRT2-overexpressing cells are hyperphosphorylated. Saos2 cells (A) and a stable transfectant overexpressing SIRT2 (clone 4 in panel B) were synchronized by double-thymidine block and subsequently treated with nocodazole or Colcemid. The DNA content of synchronized cells was assayed by flow cytometry to confirm the effectiveness of cell cycle blocks. The percentage of cells with a 2N (G₁ and S phase) or 4N (G₂ and M phase) DNA content is shown. Cell lysates were harvested at each step during synchronization and immunoblotted with SIRT2 peptide antibody. Arrows point to the phosphorylated isoforms. The arrow with an asterisk indicates λPPase-insensitive isoforms of SIRT2. UT, untreated, unsynchronized cells; T, cells harvested after the double-thymidine block; N, cells subsequently treated with nocodazole; C, cells treated with Colcemid after thymidine treatment. Blockade in M phase (Colcemid) results in a more pronounced shift of SIRT2 to the phosphorylated isoform in Saos2 cells.