Cdc14 regulates Bem3 phosphorylation. (A) Western blot of Bem3-9myc in wild-type cells during a synchronous mitosis. Cells were released from α factor and aliquots were taken at indicated times. Alpha factor was added back at 80 min to prevent the cells from starting a new cell cycle. A graph of the corresponding timing of Cdc14 nucleolar release is shown below (n = 100 cells per time point). (B) Western blot images of Rga2-9myc in wild-type cells undergoing synchronous mitosis. A graph of the corresponding timing of Cdc14 release is shown below (n = 100 cells per time point). (C) Western blots of coimmunoprecipitated 3HA-Cdc14(C283S) and Bem3-9Myc. Pgk1 serves as a loading control. (D) Western blot of Bem3-9myc in wild-type cells released from a metaphase arrest at 37°C. Cells were released from α factor into a metaphase arrest by depleting Cdc20 using a methionine-repressible promoter. Cells were incubated in methionine medium for 2 h at 25°C, and then washed and released in media lacking methionine to resume cell cycle progression at 37°C. Aliquots were taken at indicated times. A graph of cell cycle progression is shown below. Cell cycle progression was assessed by monitoring the mitotic spindle with GFP-Tub1 (n = 100 cells per time point). (E) Western blot of Bem3-9myc in cdc14-1 cells released from a metaphase arrest at 37°C (n = 100 cells per time point). Cells were processed as in D. (F) Proposed model of GTP-Cdc42 (magenta) polarity and bud localization during the cell cycle with microtubules (green), spindle pole bodies (SPBs; black), and septins (blue).