Net1 hyperphosphorylation is reduced in poor carbon. (A and B) NET1-6xHA (A) or NET1^∆328-3xHA (B) cells grown overnight in YPD or YPG/E were released from a G1 phase arrest into a fresh medium at 25°C. Samples were collected at the indicated time points to assay for Net1 and Clb2 by western blot. An anti-Nap1 antibody was used for a loading control. Alpha factor was added back to the cultures 60 min after release to prevent a second cell cycle. (C and D) Samples from multiple biological replicates of the experiment shown in A were analyzed to determine the percentage of cells undergoing bud emergence (C) and to measure median cell size with a Coulter Counter (D). (E and F) Samples from multiple biological replicates of the experiment shown in A were analyzed by immunofluorescence to determine the percentage of cells with short metaphase spindles or long anaphase/telophase spindles, and the percentage of cells that showed release of Cdc14 from the nucleolus. (G) Examples of Cdc14 localization during metaphase and anaphase for cells growing in rich or poor carbon. Error bars indicate SEM for three biological replicates. Source data are available for this figure: SourceData F3.