Clb2db does not inhibit Cdc6 by preventing Cdc6 accumulation. Metp-(HA)6-CDC6 GAL-clb2db (yJL1172, lanes 2–10) and Metp-(HA)6-CDC6 (yJL1173, lanes 11–19) cells were subjected to induction protocol I as depicted in Fig. 1B but were not released from the arrest so that (HA)6-Cdc6 accumulation (arrowhead) could be monitored by Western blot analysis (lanes 2, 3, 7, and lanes 11, 12, 16). Thirty and sixty minutes after induction of the (HA)6-Cdc6, 2 mM methionine was added back to a portion of the cultures to repress (HA)6-CDC6 expression; proteins levels were monitored at 15 min intervals by immunoblotting (arrowhead, lanes 4–6, 8–10, 13–15, 17–19). Lane 1, CDC6 (YJL312) cells grown in SCR-Leu. Sec61 was immunoblotted as an internal loading control (arrow).