Fig. 7. Drosophila Nemo-mediated phosphorylation inhibits the nuclear accumulation of Mad and MadS25A shows receptor-independent nuclear localization. COS-7 cells were transfected with (A) T7-Mad; (B) T7-Mad and HA-Tkv^QD (constitutively active form); (C) T7-Mad, HA-Tkv^QD and Flag-Nemo; (D) T7-Mad, HA-Tkv^QD and Flag-NemoK69M (kinase-dead); (E) T7-MadS25A. Immunostaining was preformed using anti-T7 and anti-HA antibodies to indicate the localization of T7-Mad (left-hand column) and expression of HA-Tkv^QD (center column). DAPI staining was also performed prior to mounting (right-hand column). Expression of Nemo (C) can inhibit the Mad nuclear accumulation that occurs upon Tkv signaling (B). Expression of kinase-dead Nemo does not affect Mad localization (D). (E) Mutation of the Nemo target site renders MadS25A constitutively nuclear even in the absence of receptor activation. (F,G) In vivo consequences of en-Gal4 expressing UAS-MadS25A (G) are very mild compared with wild-type UAS-Mad (F).