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Fig. 4. Tou physically interacts with Chip. (A) Structural features of the Chip
proteins used in this study: the N-terminal homodimerization domain (DDT) of
Chip (NTChip; black box) and the C-terminal LIM-interacting domain
(LID) of Chip (CTChip; grey box). (B) Tou interacts with Chip in
yeast through the N-terminal homodimerization domain. Expression vectors
encoding the unfused LexADBD (-) or the LexADBDChip, the
LexADBDNTChip, the LexADBDCTChip
were introduced in L40 cells together with the unfused VP16AD (not
shown) or VP16ADTouA. Protein extracts made from L40 transformants,
grown in liquid medium, were assayed for ß-galactosidase activities,
which are expressed as in Fig.
3B. (C) Tou interacts with Chip in transfected cells. The layout
is as in Fig. 3C. The flagged
Chip is detected with the M2 antibody whereas the B10-tagged proteins
(B10-Chip, B10-NTChip, B10-CTChip) are recognized by the
B10 antibody. (D) Tou directly interacts with Chip in vitro. (D)
Autoradiographs of SDS-PAGE gels from representative affinity chromatography
experiments performed with GST control beads (lane 2) and GST Chip beads (lane
3) and in vitro translated 35S proteins as indicated on the left.
One-tenth of the 35S input is shown in lane 1. Luciferase was used
as a negative input. Experiments were performed three times and 50-fold more
35S labelled TouA bound to GST Chip than to GST control. (E) The
DDT domain of Tou mediates interaction with Chip. Expression vectors encoding
the unfused LexADBD (not shown) or the LexADBDChip were
introduced into L40 cells together with the unfused VP16AD (not
shown) or the VP16ADTouA, VP16ADTouC,
VP16ADTouD, VP 16ADTouL, VP16ADTouM or
VP16ADTouN. Protein extracts made from L40 transformants, grown in
liquid medium, were assayed for ß-galactosidase activities (expressed as
in B).
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