(A) Lgl is underphosphorylated in
aurA mutants. Larval brain extracts of the indicated genotypes were analyzed.
(B) AurA induces the phosphorylation of Lgl by activation of aPKC. Immunoprecipitate (IP) from
aurA37/Ac-3 brains was incubated with ATP and recombinant AurA as indicated.
(C) ClustalW alignment of the AurA phosphorylation site (red) in Par-6 orthologs. Residues identical or similar to the
Drosophila protein are colored magenta and blue, respectively. The bottom line shows the AurA consensus ();

denotes any hydrophobic residue.
(D) Constructs used in the kinase assay.
(E) AurA phosphorylates Par-6 on Ser34 in vitro. Recombinant proteins were incubated with [
32P]ATP and recombinant AurA as indicated. The gel was Coomassie-stained (CBB), followed by autoradiography (
32P). The arrowhead indicates autophosphorylated AurA. Autoradiographs were quantified by summing the signal of the full-length bands and normalizing for the signal of Par-6
WT (set to 1). Averages and standard deviations are shown (n = 4).
(F) AurA phosphorylates Par-6 on Ser34 in vivo. Phosphospecific antibodies directed against Ser34 were used to immunoprecipitate p-Par-6 from brains of the indicated genotypes. A second round (#2) of immunoprecipitation from the supernatant confirms the depletion of p-Par-6. To avoid the overlapping IgG signal and to control for the specificity of the antibodies, extracts from wild-type animals and from
par-6Δ226 mutants complemented by a genomic rescue construct expressing Par-6-GFP (
par-6GFP) were used.