Fig. 3. Hob1 localization to sites of cell growth and division requires Nak1. (A) IRG2 (hob1-GFP) cells were grown to mid-log phase in selective medium, stained with calcofluor (left) or TRITC-phalloidin (right) and examined by fluorescence microscopy; panels on the right are enlarged. DY201 (cdc10 hob1-GFP) and DY202 (cdc25 hob1-GFP) cells were grown at 25°C to log phase and then shifted to 36°C for 4 hours to block cells before and after NETO, respectively (bottom, cdc10 and cdc25). (B) IRG7 (hob1-GFP nmt1-nak1) cells were grown in the absence (+Nak1) or presence (–Nak1) of 100 µg ml^–1 thiamine for 18 hours to repress endogenous nak1 expression. IRG7 cells expressing Nak1^T171A and Nak1^1-562 from adh1 promoter expression plasmids were also grown to repress endogenous nak1 expression (top right). WIR1 (hob1-GFP nmt1-wsp1) cells were grown in the absence (+Wsp1) or presence (–Wsp1) of 100 µg ml^–1 thiamine for 18 hours to repress endogenous wsp1 expression.