Fig. 6. Drosophila Nemo binds to and phosphorylates serine 25 in the MH1 domain of Mad. (A) pXJ-Flag-nemo and pCMV-T7-mad were co-transfected into HEK293 cells. Cell lysates were immunoprecipitated with anti-Flag, anti-T7 or IgG (control). Immunoblotting was performed with anti-Flag and anti-T7 antibodies. (B) Nemo phosphorylates Mad and autophosphorylates. HEK293 cells were transfected with expression vectors as indicated. Immunoprecipitated complexes with indicated antibodies were subjected to in vitro kinase assays and analyzed by autoradiography. The immunoprecipitates were also immunoblotted with the indicated antibodies to confirm loading. (C) Schematic of the full-length Mad protein showing the MH1, MH2 and linker domains, as well as the site of the nuclear localization sequence (NLS). Potential Nemo phosphorylation sites are each indicated directly above the protein structure as a numbered S residue, followed by proline (P). The constructs shown beneath were generated to identify residues that are phosphorylated by Nemo. (D) In vitro kinase assays performed with wild-type Mad, Mad 4SA, Mad AAVA, Mad-MH1 and MadS25A demonstrate that Nemo specifically targets serine 25, and that Nemo autophosphorylates. (E) Immunoblot of cell extracts used in kinase assays showing relative expression levels of these proteins.