Figure 4

The rapid G₂/M transition of HeLa cells over-expressing TSPY is associated with an early degradation of the mitotic cyclin B1. HeLa Tet-off cells harboring the vector alone (A) and TIG-TSPY construct (B) were synchronized at G₁/S, released into S phase, harvested and analyzed with western blotting with various antibodies at 2-hour intervals for 22 hours. The mitotic cyclin B1 is synthesized normally in late S phase and G₂ and is rapidly degraded before the cell exits mitosis. In cells over-expressing EGFP in the vector alone, the levels of cyclin B1 increased at 6 hours after the G₁/S release, peaked at 10 hours and gradually decreased until 14–16 hours (A, top row) while in those over-expressing both EFGP and TSPY, the levels of cyclin B1 increased gradually at 2 hours after the G11/S release, peaked at 10 hours and rapidly reduced thereafter (B, top row). The filters were re-probed with tubulin antibody (A, bottom row; B, middle row) showing relatively even loading of the samples. TSPY was uniformly expressed and detected in samples of cells over-expressing this transgene (B, bottom row).