Fig 8. Hpo competes with Herc4 for Sav binding and antagonises Herc4-mediated ubiquitylation of Sav.

(A) Hpo reduces Herc4-induced ubiquitylation of Sav. Sav ubiquitylation assay was performed using Myc-tagged Sav co-expressed with either Hpo, Herc4 or Hpo and Herc4 combined. Ubiquitylation assay was performed in the presence of 5mM NEM to block deubiquitylating enzyme activity. (B) Binding of Sav to Herc4 is prevented by active Hpo kinase (compare left with right lane), while kinase-dead Hpo has no effect (compare left and middle lanes). Herc4/Sav co-IP experiments were performed in the presence of a control protein (GFP), wild-type or kinase dead version of Hpo (HpoT195A). (C) herc4 RNAi rescues the destabilizing effect of hpo RNAi on Sav protein levels. S2 cells expressing Myc-tagged Sav (and GFP-Flag as a control) were treated with the indicated combinations of dsRNA.