Fkh2 is differentially phosphorylated between yeast and hyphal growth.

<p>A) Early G1 cells expressing Fkh2-YFP were collected by elutriation and re-inoculated into yeast growth conditions. Samples were taken for αGFP Western blot to observe Fkh2 phosphorylation and microscopy to follow cell cycle progression via budding and DAPI stained nuclei (n = 50) Note YFP is recognised by the αGFP monoclonal antibody; αCdc11 was used as a control for equal loading. B) Early G1 cells expressing Fkh2-YFP and Cdc12-mCherry were collected by elutriation and re-inoculated into hyphal growth conditions. Samples were taken as above, with cell cycle progression followed by monitoring septin ring formation and nuclear migration/division (n = 50). C) Confirmation of Fkh2 phosphorylation by phosphatase treatment. 80 min yeast and 40 min hyphae samples were taken and lysates treated at 30°C for 1 h with/without Lambda-phosphatase (NEB) and then resolved by 7% 1D PAGE. D) Fkh2 phosphorylation early on hyphal induction. Samples were taken at the indicated time points after hyphal induction and resolved by 1D PAGE as previously mentioned. In <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.g001" target="_blank">Figs. 1B–D</a> αPSTAIRE was used as the loading control. E) Fkh2-YFP was isolated from cells in the culture conditions and times indicated and fractionated by 2D gel electrophoresis. Note the region of darkening at the acidic edge of the gel is where the sample was applied and does not come from Fkh2. An intensity profile is shown above each autoradiograph. In this and subsequent figures the profile was scaled to give maximum height to the maximum peak in the informative part of the gel. Where necessary some values from the non-specific part of the gel were omitted. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.g001" target="_blank">Fig. 1E</a> is shown with an independent replicate in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004630#ppat.1004630.s002" target="_blank">S2 Fig.</a>