Fig 6. Ysc84 and Rvs167 bind to a common site on Las17.

(A) Yeast two-hybrid analysis was carried out in which the polyproline domain of Las17 was carried on an activation plasmid and the Ysc84 or Rvs167 SH3 domains were expressed on the Gal4 binding domain (bait) plasmid. Mutations of Las17 carrying P387A and P388A mutations were used to reveal inhibition of binding of both SH3 domains. (B) Membrane dot blots carrying overlapping 12mer peptides of Las17 over the region 373–406 were incubated with GST fused SH3 domains of Ysc84 and Rvs167 or with GST alone. Proteins were detected by western blotting for GST.