Fig. 1. Par-1 phosphorylates Exu in vitro and in vivo. (A) In vitro translated and ³⁵S radiolabelled Exu protein subjected to SDS-PAGE and autoradiography. Exu protein incubated in kinase buffer alone migrates as three bands consisting of a major band at 60 kDa and two weaker protein bands that migrate more slowly (lane 2). As phosphorylation usually reduces the electrophoretic mobility of a protein, the presence of the two weak bands of Exu suggests that the protein is phosphorylated by kinases present in the in vitro translation extract. The absence of the slowly migrating protein bands after 2 hours treatment with lambda phosphatase shows that Exu is phosphorylated (lane 3). Phosphorylation is dramatically increased after incubation with Par-1 kinase, as revealed by the absence of the 60 kDa fast migrating band and the appearance of a slowly migrating protein doublet at 66 kDa (lane 1). (B) Western blot of ovarian extracts of females of the indicated genotypes probed with an antibody against Exu (upper panel) or Tubulin (lower panel) as a loading control. par-1^W3 is a null allele, and par-1^9A and par-1⁵⁷⁴ are hypomorphic alleles. par-1 overexpression in ovaries was achieved by placing par-1 under the control of the Gal4 UAS system and expressing a UAS-par-1 transgene in the germline using the nanosGal4:VP16 driver.