Fig. 6. The phosphorylation state of Wee1p is not affected by loss of flp1p function. (A) A metaphase block- and-release experiment of nda3-KM311 wee1_3HA (control) and nda3-KM311 wee1_3HA flp1 (otherwise) isogenic strains. Cells were synchronously released from an nda3 block (6 hours at 20°C), samples were taken at the indicated (in minutes) intervals to analyse Wee1p protein phosphorylation by shift mobility after western analysis with -Ha monoclonal antibody. The plot of septated cells (Septation Index) is shown. (B) Cdc25p localises at the nucleolus when flp1⁺ is overexpressed. Photomicrographs of nmt1: flp1⁺ or nmt1: flp1⁺ myc₁₂-cdc25 cells induced for the expression of Flp1 for 18 hours. (C) Overexpression of flp1⁺ induces Cdc25p dephosphorylation. Expression of Flp1 was induced in an nmt1: flp1⁺ strain by removal of thiamine from the medium, samples were taken at indicated intervals and processed for western analysis and immunofluorescence (see B).