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Fig. 3. Syph interacts with DE-cadherin and several MT-associated proteins
through its FERM domain. (A) 35S-labelled in vitro
translated (IVT)-Syph C-terminal tail (input) binds to GST-cadICD,
-Katanin, -aPKC, -Milt and -EB1. (B) DE-cadherin was immunoprecipitated
from embryo lysate with an anti-Syph antibody, but not when primary antibody
was omitted. (C) Diagram of the Syph protein fragments and substitution
point mutations used to map protein-protein interactions. (D,E)
GST-cadICD binds 35S-labelled IVT-Syph tail, -Syph t3,
and the -L1 and -L3 regions of the Syph FERM domain. GST-cadICD
binding maps to the L1a3 fragment within the L1 region and the L3b fragment
within the L3 region. (F,G) Substitution point mutations in the
L1 or L3 regions do not selectively inhibit cadICD binding to Syph.
GST-cadICD and -Milt bind to 35S-labelled IVT-L1, -L3,
-LGVE*, -QEF* and -HWS*, but show
significantly reduced binding to IVT-IVQG*, -DAFT*,
-STR* and -DMK*. GST-Katanin binds to IVT-L1, -L3, and
all of the mutant fragments except IVT-DMK*. GST-aPKC binds to
IVT-L1, -L3, and all the mutant proteins tested. EB1 binding does not map to
the L1 or L3 regions and serves as a negative control. (H) Diagram of
DE-cadherin protein and the protein fragments used to map its interaction with
Syph. JMD: juxtamembrane domain, CBD: catenin binding domain; TMD:
transmembrane domain. (I) 35S-labelled Syph-tail and -t3
proteins bind to GST-cadICD and -cad-D. (J-J'')
Computer model of the Merlin FERM domain crystal structure. The regions
corresponding to L1 and L3 in Syph are shown in green and blue, respectively,
and lie on the same surface of the protein as shown in side (J) and topdown
(J') views. (J'') The position of the point mutations in L1 and L3
that disrupt Syph binding to cadICD are shown in purple and pink,
respectively.
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