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Fig. 5. Bar interacts with the Chip and Ap proteins. (A) Representation of
different domains in Chip and deleted Chip proteins. Chip contains a proline
and glutamine rich (PQ rich) region at the amino-terminal end, followed by a
Dimerisation Domain (DD). The LIM interaction domain (LID) is located at the
carboxyl-terminal end. The Other Interaction Domain (OID) appears between
amino acids 439 and 456, and mediates the interaction with Bicoid. The
Chip
LID protein lacks the LIM interaction domain, and the
ChipOID lacks the OID domain. (B) Sample western blots of the affinity
chromatography experiments using leg disc extracts; Gst-Chip fusion proteins
and beads used are indicated at the top of the lanes, and the different
antibodies used for immunodetection are indicated on the left. The
‘Gst’ and ‘Beads’ lanes show the lack of protein
retained by beads with the Gst protein alone, and by the Gluthathione-agarose
beads alone, respectively. Other lanes on the top row show an 
62 kDa band
in the anti-Bar western blot, corresponding with the predicted size of Bar.
Bar is able to interact with Chip, and with the Chip LID- and Chip OID-deleted
proteins, but it does not interact with Gst or with beads alone. Similarly, in
the middle row a 46 kDa band is detected in the anti-Lim1 western blot,
showing that Chip interacts with Lim1. However, a decrease of signal of this
band is detected in the ChipLID lane, as has also been found with Ap
(Torigoi et al., 2000),
corroborating that the LID is crucial for the interaction between Ldb and
LIM-HOM proteins. The lack of the OID domain does not affect this interaction.
Finally, in the bottom row the western blot shows that Al interacts with Chip.
An 40 kDa band corresponding with the predicted size of Al is detected. A
decrease of the signal is observed in the ChipLID lane and the signal
is almost undetectable in the ChipOID lane. Thus, both protein domains
are necessary for the proper binding of Al. (C) Representation of the protein
domains in Ap and Ap-LIM proteins. The Ap protein contains two LIM domains at
the amino-terminal part of the protein followed by a homeodomain. The Ap-LIM
protein consists of the amino-terminal end containing the LIM domains. (D)
Western blot carried out similar to that shown in B, but with Gst-Ap
constructs. The same 62 kDa band was detected using the anti-Bar antibody. Bar
interacts with Ap and Ap-LIM, as well as with Chip, but it does not interact
with Gst or with beads alone. The increase of signal in the Ap-LIM lane in
comparison with in the Ap and Chip lanes is due to the higher molarity of
Ap-LIM protein loaded in comparison with Ap and Chip proteins.
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