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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-TkvQD (constitutively active form);
(C) T7-Mad, HA-TkvQD and Flag-Nemo; (D) T7-Mad,
HA-TkvQD 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-TkvQD (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).
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