Cdc14 localization and Cdc5 degradation in wild-type cells and MEN mutants. (a–f) cdc15-as1 (Ry1132; a, c, and e) and wild-type (Ry278; b, d, and f) cells were arrested in G1 with α-factor in YEPD at 23°C and released in fresh YEPD media supplemented with the Cdc15-as1 inhibitor (1NM-PP1 analogue 9; Bishop et al., 2001). (a and b) At the indicated time points, samples were taken to determine the percentages of metaphase spindles (closed squares), anaphase spindles (closed circles), and cells with Cdc14 released from the nucleolus (open circles). Examples of Cdc14 localization for the 105-min time point are shown. (c and d) Cdc14 is shown in red, tubulin in green, and DAPI in blue. Bars, 3 µm. (e and f) For the entire time course, we analyzed Clb5, Clb2, Cdc5, and Pgk1 protein levels. Pgk1 protein was used as an internal loading control in immunoblots. Black lines indicate that intervening lanes have been spliced out. Size markers on the sides of the gel blots indicate kilodaltons.