Analysis of candidate Net1 kinases in vivo. For all experiments, cells were grown in YPD medium and released from a G1 phase arrest into fresh YPD media containing benomyl. Inhibitor was added at the times indicated by the arrow. Samples were then taken at the indicated time points to assay for NET1^∆328 and Clb2 by western blot. (A)NET1^∆328-3xHA and NET1^∆328-3xHA cdc5-as1 cells were released from G1 phase at 30°C. 10 μM CMK was added 30 min after release. (B)NET1^∆328-3xHA and NET1^∆328-3xHA cdc15-as1 cells were released from G1 phase arrest at 25°C. 10 μM 1-NA-PP1 was added 40 min after release. (C)NET1^∆328-3xHA cells were released from G1 phase arrest at 25°C. The culture was split into two aliquots and 0.22 μM rapamycin was added to one aliquot 90 min after release. (D)NET1^∆328-3xHA and NET1^∆328-3xHA sch9-as1 cells were released from G1 phase at 25°C. 5 μM of 1-NM-PP1 was added 30 min after release. Source data are available for this figure: SourceData FS1.