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, Antp^AAAA (AHA), hs>bip2, Antp (Y), hs>bip2, Antp^AAAA (A), and wild type (WT), as a control.