Fig. 1. Interaction of Sla1p with proteins involved in actin dynamics. (A) GST-Sla1p was overexpressed in yeast as described in Materials and Methods and purified on glutathione sepharose beads following ion exchange chromatography. Yeast extracts were incubated with either GST-Sla1p beads or with glutathione beads alone. Beads were spun down, washed and bound proteins eluted in SDS sample buffer. Analysis was by western blotting using antibodies as marked. (Lanes: FT, flow through; W3, third wash; B, bound). (B) Further evidence of the Sla1p-Abp1 interaction was demonstrated using immunoprecipitation. Yeast cell extracts from a strain expressing Sla1-HA (KAY355) were incubated with protein-A sepharose bound with anti-Abp1p antibodies. After washing, the bound proteins were separated by SDS-PAGE and transferred to PVDF. The blot was probed with antibodies to Abp1p or HA to detect Sla1p, Srv2p and Sac6p. E, extract; W3, third wash; B, bound.