Yeast drug treatments were performed in YPD media at the following concentrations: 400 μg/ml 1,10 phenanthroline monohydrate (Sigma 161-0158, dissolved in ethanol), 10 μg/ml thiolutin (Santa Cruz SC-200387, dissolved in DMSO), 1 mM 1-naphthalene acetic acid (Sigma N0640, dissolved in 85% ethanol), 20 μg/ml doxycycline (Sigma D9891, dissolved in 50% ethanol), 5 μM 1-Naphthyl PP1 (Sigma CAS 221243-82-9, dissolved in DMSO), 25 μM trichostatin A (dissolved in DMSO), 10 μM α factor (Sigma custom synthesized peptide, WHWLQLKPGQPMY, dissolved in 100 mM sodium acetate, pH=5.2). mESCs were treated with Actinomycin D at 25 μg/ml (Sigma CAS 50-76-0, dissolved in DMSO).
Whwlqlkpgqpmy
Manufactured by Merck Group
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3 protocols using whwlqlkpgqpmy
1
Yeast Drug Treatment Protocols
2
Synchronizing yeast cells using alpha-factor
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Cells were grown at 30°C in YEP (1% yeast extract, 2% bactopeptone) medium supplemented with 2% glucose. For synchronisation, overnight cultures were diluted to OD600=0.1, grown for 3 h and arrested by adding 3 µg/ml α-factor (Sigma, custom peptide WHWLQLKPGQPMY) twice, with a 60 min interval. Cells were released by washing with YEP, resuspending in YEP, and incubating at 30°C. To rearrest the cells in the next G1, 3 µg/ml α-factor was added again to the cultures 40 min after the release.
3
Cell Cycle Synchronization Protocol
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Cells were grown at 30°C in YEP (1% yeast extract, 2% bacto-peptone) medium supplemented with 2% glucose (YEPD) unless otherwise stated. For G1 arrest, overnight cultures were diluted to OD600 = 0.1 in fresh media, grown for 2.5 hours and arrested by adding 3 µg/ml ɑ-factor (Sigma, custom peptide WHWLQLKPGQPMY) every 60 min for 120 min. Cells were released by washing away ɑ-factor with 1x culture volume of YEPD and resuspending in 1x culture volume of YEPD, and further grown at 30°C. For metaphase arrest, PGal1-Cdc20 cells were grown in YEP supplemented with 2% raffinose and 2% galactose, and switched to YEPD for 120 min.
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