Net1 hyperphosphorylation occurs before mitotic exit. (A) NET1-6xHA cells growing in YPD were released from a G1 arrest at 25°C into YPD or YPD containing benomyl. Samples were collected at the indicated time points to assay Net1 phosphorylation and Clb2 protein levels by western blot. Alpha factor was added back to the cultures at 60 min after release to prevent a second cell cycle. (B)NET1^∆328-3xHA cells growing in YPD were released from a G1 arrest at 20°C into YPD or YPD containing benomyl. Samples were collected at the indicated time points to assay Net1 phosphorylation and Clb2 protein levels by western blot. Alpha factor was added back to the cultures at 60 min after release to prevent a second cell cycle. (C)NET1^∆328-3xHA GAL1-CDC20 cells were grown overnight to log phase in YPGal medium at room temperature and were released from a G1 phase arrest into YPGal or YPD at 25°C. Samples were collected at the indicated time intervals to assay for NET1^∆328-3xHAand Clb2 by western blot. In all panels, the Net1 blots and the Clb2 blots are from the same samples, so the Net1 blots serve as a loading control for the Clb2 blots. Source data are available for this figure: SourceData F2.