Fig. 9. Hob1 overexpression or nak1 repression inhibits Wsp1-mediated F-actin formation in vitro. (A) NTA-agarose beads were either coated with Wsp1-HAHis₆ by incubation in extracts from yeast expressing this protein or incubated in control yeast lysates (control). They were then incubated with fluorescent Alexa-568/actin and wild-type (SPU) cell extract (WT), wild-type extract in the presence of 10 µM LatA (+LatA) or XB-200 buffer alone (no extract). The ability of the different extracts to stimulate Wsp1-mediated F-actin formation was observed by the presence of fluorescent F-actin halos around the beads. A proportion of the NTA-agarose beads were boiled and immunoblotted with the anti-HA (12CA5) antibody to detect Wsp1-HAHis₆ bound to the beads. (B) NTA-agarose beads were similarly incubated in control extracts or extracts from yeast expressing Wsp1-HAHis₆ (Wsp1), Wsp1-HAHis₆ and myc-Hob1 (+Hob1), or Wsp1-HAHis₆ and myc-Hob1^1-281 (+Hob1^1-281), and were assayed for the formation of F-actin halos in Alexa-568/actin in the presence of wild-type extract. The presence of Wsp1-HAHis₆ (shown on the right) and myc-Hob1 or myc-Hob1^1-281 (not shown) were confirmed by western blots. (C) Wsp1-HAHis₆-coated beads were assayed for F-actin formation in the presence of cell extracts derived from nak1-repressed (TYH1) cells containing a control vector (–Nak1) or expressing either Nak1 (+Nak1) or Nak1^T171A (+Nak1^T171A) grown in the presence of 100 µg ml^–1 thiamine. No F-actin formation was observed in beads coated using control extracts (derived from the wild-type SPU strain, not shown).