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Fig. 6. Su(fu) protein is phosphorylated in response to Hh overexpression. (A)
Western blot of extracts from imaginal wing discs probed with anti-Su(fu)
antibody. BAP 111 was used as a loading control. Lane 1, extract from
wild-type discs; lane 2, extract from Hh overexpressing discs; lane 3, extract
from Cos2-overexpressing discs; lane 4, extract from discs expressing S182N;
lane 5, extract from discs expressing S182T; lane 6, extract from discs
overexpressing both Hh and Cos2 simultaneously; lane 7, extract from discs
overexpressing both Hh and S182N simultaneously. Overexpression of proteins
was achieved in each case by using the 71B Gal4 driver. Arrowheads indicate
the migration of singlet and doublet bands recognized by the Su(fu) antibody.
The Su(fu) doublet appears only in those lanes in which Hh has been
overexpressed. (B) Treatment of wing disc lysates with the lambda phosphatase
enzyme, which removes phosphates from both Ser/Thr and Tyr, shifts the doublet
Su(fu) band to a single band that co-migrates with Su(fu) from wild-type
lysates. Lane 1, extract from 71B Gal4 wild-type discs, showing a single band
for Su(fu); lane 2, the Su(fu) doublet is seen in lysates from
Hh-overexpressing discs; lane 3, treatment of 71B Gal4 wild-type lysates with
lambda phosphatase does not change the migration properties of Su(fu), compare
lane 3 with lane 1, suggesting that the majority of Su(fu) does not exist in a
phosphorylated state in wing imaginal discs. Lane 4, treatment of
Hh-over-expressing lysates with lambda phosphatase shifts the Su(fu) doublet
(Lane 2) to a singlet (arrowheads). Dsh, Dishevelled protein, recognized by
Rabbit anti-Dsh (gift from Karl Willert and Roel Nusse), was used as a control
as it normally exists as a hyperphosphorylated protein (lane 1). The
successful dephosphorylation of Dsh can be seen by a change in its migration
as multiple bands (lanes 1 and 2) to a single major band (lanes 3 and 4).
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