Fig. 4. Mapping and mutation of Exu phosphorylation sites. (A) Position of the two potential 14-3-3 binding sites (red) in Exu protein (grey bar). Sequences of site A (amino acids 434-440) and B (amino acids 453-459) and of the corresponding mutations (A1-A6 and B1-B3) are shown below. (B-D) Autoradiographic images of SDS polyacrylamide gels showing in vitro translated and radiolabelled wild-type or mutant Exu proteins after incubation with Par-1. Mutations in site A and B are indicated above the lane. (E) Western blot of ovarian extracts probed with an antibody against Exu (upper panel) or Tubulin (lower panel) as a loading control. Exu protein was expressed from transgenes using the endogenous exu promoter in an exu-null background (exu^XL/exu^VL). Transgenes encode either wild-type or mutant Exu protein. The nature of the mutation is indicated above the corresponding lane. The first lane shows extracts from exu null mutant ovaries expressing Exu-GFP protein, in which all Par-1 phosphorylation sites are mutated (GFP A6B3). This mutant protein runs in a single band, indicating absence of phosphorylation. The second lane shows extracts from wild-type ovaries expressing Exu-GFP. Exu-GFP is phosphorylated and migrates as a triplet around 100 kDa while untagged Exu migrates around 66 kDa.