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Fig. 2. Drosophila BIP2 interacts directly with ANTP via the YPWM
motif. (A) Schematic of the Antp constructs used fused to
the LexA DNA-binding domain. LexA-YPWM-HD consists of the YPWM motif, the
linker region and the HD with 
-helices 1-3 (H1-H3). LexA-YPWM-N-term
consists of the YPWM motif, the linker and the N-terminal arm of the HD.
LexA-AAAA-N-term is the same construct as LexA-YPWM-N-term with the YPWM motif
substituted by four alanines. LexA-YPWM-consists of the YPWM motif and the
linker region. LexA-AAAA- is the same construct as LexA-YPWM- with the YPWM
motif substituted by four alanines. LexA- is an empty vector. (B) X-gal
filter-lift experiment. (C) Relative β-gal activity. The
β-gal experiment was repeated four times independently using three
samples of each interaction tested. (D) Glutathione-S-transferase (GST)
pull-down experiments. The ANTP-YPWM-HD-GST fusion protein (amino acid
279-356) was produced in E. coli and purified with glutathione
sepharose beads. The BIP2-235 protein (amino acids 853-1088) was produced in a
rabbit reticulocyte lysate and labelled with [35S]-methionine. The
BIP2 protein domain found in the yeast two-hybrid screen (BIP2-235) is also
able to interact with ANTP-HD fused to GST in vitro. The synthesis of the
BIP2-235 protein gives three bands, likely to be due to different methionine
start codons used for protein synthesis by the reticulocyte lysate. (E)
Co-immunoprecipitation of ANTP and BIP2. The BIP2 protein was tagged with the
hemaglutinin (HA) epitope and immunoprecipitated with an anti-HA antibody.
Co-immunoprecipitated ANTP protein was detected by using a mouse monoclonal
anti-ANTP antibody. Upon mutating the YPWM motif of ANTP, ANTP is not
co-immunoprecipitated with BIP2-HA, unlike the wild-type ANTP protein. The
larvae used were hs>bip2-HA, Antp (YHA), hs>bip2-HA,
AntpAAAA (AHA), hs>bip2, Antp (Y), hs>bip2,
AntpAAAA (A), and wild type (WT), as a control.
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