1. Volume 2, Issue 6, Pages 709-718 (December 1998)
The Phosphatase Cdc14 Triggers Mitotic Exit by Reversal of Cdk-Dependent Phosphorylation 
Rosella Visintin, Karen Craig, Ellen S Hwang, Susanne Prinz, Mike Tyers, Angelika Amon 
Molecular Cell 
Volume 2, Issue 6, Pages (December 1998)
DOI: /S (00)

2. Figure 1 The Terminal Phenotype of cdc14-3 Mutants
cdc14-3 mutants (A1263) were arrested in G1 with α factor pheromone (5 μg/ml). When arrest was complete (after 2.75 hr), cells were released into medium lacking pheromone at 37°C. Samples were withdrawn at the indicated times to analyze the amount of Pds1-Ha, Ase1, Clb2, Sic1, and Cdc28 protein (A), Clb2-associated kinase activity (B), SIC1 and CDC28 RNA (C), percentage of metaphase and anaphase spindles (D), and DNA content (E). Cdc28 protein and CDC28 RNA were used as internal loading controls in immunoblot and Northern blots, respectively.
Molecular Cell 1998 2, DOI: ( /S (00) )

3. Figure 2
cdc14-3 Mutants Are Defective in Clb2 Degradation and Sic1 Stabilization
(A) Wild-type cells (K699) were arrested at 37°C for 2.5 hr in the presence of 15 μg/ml nocodazole. cdc14-3 (A794) and cdc15-2 (K1993) mutants (cdc15-2 is another mutant that arrests in anaphase) were incubated in the absence of nocodazole. Cells were then pulse labeled with [35S]methionine for 7 min followed by chase using 2 mM cold methionine and 1 mg/ml cycloheximide (t = 0).
(B) Wild-type cells carrying a GAL-SIC1 fusion (A701) were arrested at 37°C in the presence of 15 μg/ml nocodazole (Noc) or 10 mg/ml hydroxyurea (Hu) or 5 μg/ml α factor. cdc14 (A865) and cdc4 (A1345) mutants were arrested by shifting cells to 37°C. After 3 hr, the half-life of Sic1 was measured as described in Experimental Procedures. Kar2 protein was used as an internal loading control in immunoblots. In parallel, samples were withdrawn to determine cell cycle position by DNA content measurements.
Molecular Cell 1998 2, DOI: ( /S (00) )

4. Figure 3
Overexpression of CDC14 Induces Ectopic APC-Dependent Proteolysis and Sic1 Accumulation
Wild-type cells (A1015), cells carrying three copies of CDC14 under the control of the GAL1-10 promoter (A1262), and GAL-CDC14 cells carrying a cdc23-1 mutation (A1264) or a SIC1 deletion (A1311) were grown to exponential phase. Next, galactose was added (t = 0 min), and samples were withdrawn at the indicated times for 240 min.
(A) DNA content analysis.
(B) Immunoblot analysis of Clb2, Pds1-Ha, Sic1, and Kar2 protein.
(C) SIC1 and 25S rRNA.
(D) Clb2-associated histone H1 kinase activity.
Molecular Cell 1998 2, DOI: ( /S (00) )

5. Figure 4
Overexpression of CDC14 Induces APC-Dependent Proteolysis and Sic1 Accumulation in Hydroxyurea-Arrested Cells
Strains described in the legend to Figure 3 were arrested with hydroxyurea (10 mg/ml) at 23°C. After 3 hr, galactose was added (t = 0 min.), and samples were withdrawn at the indicated times to determine total levels of Pds1-Ha, Clb2, Sic1, and Cdc28 protein (A), SIC1 and 25S rRNA (B), and Clb2-associated kinase activity (C).
Molecular Cell 1998 2, DOI: ( /S (00) )

6. Figure 7
GAL-CDC14-Induced Clb Degradation and Sic1 Accumulation in cdh1, dbf2, cdc5, and cdc15 Mutants
(A) Wild-type (A1534) and cdh1Δ cells (A1532) both carrying one copy of the GAL-CDC14 fusion were arrested with nocodazole at 23°C followed by galactose addition. Time points were taken to determine the amount of Clb2, Pds1-Ha, and Sic1 protein.
(B) Left panel: CDH1-HA cells (A1576) were arrested with 15 μg/ml nocodazole (Noc). Cdh1-Ha was then immunoprecipitated and incubated in the presence or absence of calf intestinal alkaline phosphatase (CIAP), followed by immunoblot analysis using anti-Ha antibodies. Right panel, lanes 1–3: CDH1-HA cells (A1576) were arrested with 5 μg/ml α factor (α-F) or nocodazole (Noc) in the presence of galactose for 3 hr. GAL-CDC14, CDH1-HA cells (A1640) were grown in the presence of galactose for 3 hr. Lanes 4–6: CDH1-HA cells (A1576) were arrested with 5 μg/ml α factor (α-F) or nocodazole (Noc) for 3 hr at 30°C. cdc14-3, CDH1-HA cells (A1642) were arrested at 30°C for 3 hr. Cdh1-Ha was immunoprecipitated and visualized by immunoblot analysis using anti-Ha antibodies.
(C) Wild-type cells overexpressing CDC14 (A1220) were arrested with nocodazole at 37°C; cdc5-1 (A1239), dbf2-2 (A1240), or cdc15-2 (A1236) mutants carrying a GAL-CDC14 fusion were arrested at 37°C. After 3 hr, galactose was added (t = 0), and Clb2 and Sic1 protein levels were analyzed at the indicated times.
Molecular Cell 1998 2, DOI: ( /S (00) )

7. Figure 5 Effects of Overexpression of CDC14 on Sic1
(A) SIC1 deletion cells carrying an ADH-SIC1 fusion (A1397) were either grown to exponential phase (Cyc) or arrested with α factor (α-F), or hydroxyurea (Hu), or nocodazole (Noc) in medium containing galactose. GAL-CDC14, sic1Δ cells carrying the same fusion (A1412) were incubated in the presence of galactose to analyze Sic1 protein and RNA.
(B) Wild-type (A1397) and GAL-CDC14 (A1412) cells carrying SIC1 under the ADH promoter were arrested with nocodazole followed by galactose addition (t = 0). At the times indicated, samples were withdrawn to analyze the amount of Sic1 and Kar2 protein and SIC1 and CDC28 RNA.
(C) DNA content analysis of wild-type cells (A1015), GAL-CDC14 cells (A1262), and GAL-CDC14 cells lacking SIC1 (A1311) after release from an α factor–induced G1 arrest in the presence of galactose.
Molecular Cell 1998 2, DOI: ( /S (00) )

8. Figure 6 Swi5 and Sic1 Are Substrates of Cdc14 In Vitro
(A) GST-Swi5 (amino acids 496–709) or His6-Sic1 were radiolabeled with 32P as described in Experimental Procedures. Approximately 100 ng labeled GST-Swi5 or 200 ng of His6-Sic1 was incubated with the indicated amounts of recombinant GST-Cdc14CS, GST-Cdc14, or calf intestinal phosphatase (CIAP). Reactions were separated by SDS-PAGE. The Coomassie-stained gel shown in the second panel corresponds to the 32P Gst-Swi5 autoradiogram shown in the top panel. Similar results were obtained using GST full-length Swi5 (data not shown). The Western blot shown in the bottom panel corresponds to the 32P His6-Sic1 autoradiogram shown in the third panel.
(B) In vitro translated, [35S]methionine-labeled Cdc14 with or without a FLAG tag, or a temperature-sensitive Cdc14-3 or Cdc14-3-flag protein was mixed with 10 ng of recombinant Sic1-GST. Cdc14 was immunoprecipitated using an anti-FLAG M2-agarose. Immunoprecipitated Sic1 was visualized by immunoblotting; 35S-labeled Cdc14 and Cdc14-flag were visualized by fluorography.
Molecular Cell 1998 2, DOI: ( /S (00) )

9. Figure 6 Swi5 and Sic1 Are Substrates of Cdc14 In Vitro
(A) GST-Swi5 (amino acids 496–709) or His6-Sic1 were radiolabeled with 32P as described in Experimental Procedures. Approximately 100 ng labeled GST-Swi5 or 200 ng of His6-Sic1 was incubated with the indicated amounts of recombinant GST-Cdc14CS, GST-Cdc14, or calf intestinal phosphatase (CIAP). Reactions were separated by SDS-PAGE. The Coomassie-stained gel shown in the second panel corresponds to the 32P Gst-Swi5 autoradiogram shown in the top panel. Similar results were obtained using GST full-length Swi5 (data not shown). The Western blot shown in the bottom panel corresponds to the 32P His6-Sic1 autoradiogram shown in the third panel.
(B) In vitro translated, [35S]methionine-labeled Cdc14 with or without a FLAG tag, or a temperature-sensitive Cdc14-3 or Cdc14-3-flag protein was mixed with 10 ng of recombinant Sic1-GST. Cdc14 was immunoprecipitated using an anti-FLAG M2-agarose. Immunoprecipitated Sic1 was visualized by immunoblotting; 35S-labeled Cdc14 and Cdc14-flag were visualized by fluorography.
Molecular Cell 1998 2, DOI: ( /S (00) )