Zymolyase

To measure growth inhibition caused by either zymolyase or tunicamycin, cell cultures were inoculated from an overnight culture to an O.D. _{620 nm} = 0.025 in YPD medium supplemented with different amounts of either zymolyase100T (ICN Biomedicals, Inc., dissolved in Tris-HCl [pH 7.5]–5% glucose) or tunicamycin (Sigma-Aldrich, dissolved in DMSO). The assay was performed using duplicate rows of a 96-well plate and incubated overnight at 37°C. Graphs represented the percentage of growth estimated by O.D. _{620 nm} measures for each strain in YPD plus both compounds compared to similar untreated cells.

Cells were fixed in growth medium with 3.7% formaldehyde at room temperature for 30 minutes. Mitotic cells were digested in SCE (1 M sorbitol/0.1 M sodium citrate/10 mM EDTA) with 20 μg/ml zymolyase (100T, ICN Biomedicals). Meiotic cells were digested with a solution containing 50 μg/ml zymolyase and 1:50 glusulase (Perkin Elmer). Cells were mounted on poly-L lysine coated slides and proteins detected by indirect immunofluorescence.
Immunostaining of whole cells was as follows: Cdc14-Myc7 fusion protein was detected with mouse monoclonal anti-Myc 9E10 antibody (Covance) at 1:600 in PBS/1% BSA for 3 hours at room temperature, followed by anti-mouse CY3 (Jackson ImmunoResearch) secondary antibody at 1:600 for 1 hour at room temperature. Slide preparation was finished by adding mounting medium containing DAPI. Detection of tubulin was done with the rat monoclonal antibody YOL 1/34, followed by anti-rat FITC (Jackson ImmunoResearch).