CENP-A is protected by the kinetochore ensemble.

<p>(A) J108 (<i>PCK1</i>pr<i>DAD2</i>/<i>dad2</i>) was grown overnight in media expressing Dad2 (+Suc), transferred to media that repressed Dad2 (+Glu), and cells were harvested at specific time intervals as shown. Western blot analysis was performed using anti-Dad2, anti-Cse4 or anti-PSTAIRE antibodies with cell lysates prepared from these cells. Both Dad2 (left panels) and CENP-A/Cse4 (right panels) protein levels showed a gradual decrease as time of repression of Dad2 prolonged. PSTAIRE was used as the loading control. Increasing amount of protein was loaded to visualize the reduced Dad2 or CENP-A/Cse4 protein signals in these western blots. (B) Western blot analysis using anti-Cse4 and anti-PSTAIRE antibodies with cell lysates prepared from conditional mutant strains CAKS12 (<i>PCK1</i>pr<i>MTW1/mtw1</i>) and YJB12326 (<i>MET3</i>pr<i>NUF2/nuf2</i>) before (0 h) and at indicated time of incubation in non-permissive media after shift. Relative levels of Cse4 or Dad2 normalized by corresponding levels of PSTAIRE are shown.