Figure 3

The WDR domain of Tle3b binds to the C-terminus of Hmx3a but not to any canonical Tle interaction motifs. (a) Schematics of FLAG-tagged Hmx3a constructs used in these experiments. eh1A, eh1B, and WYPY indicate predicted interaction motifs for Tle3b-WDR. Numbers (top) indicate residues encompassing annotated domains or (left-hand side, bottom row) included in the C-terminal construct. (b, c) The WDR domain of Tle3b was expressed as a GST-fusion protein, purified, and incubated with either a FLAG-Hmx3a-expressing embryo lysate or a stage-matched control lysate expressing no recombinant Hmx3a. Proteins were immunoprecipitated with an anti-FLAG antibody and separated by SDS-PAGE followed by transfer to a membrane and immunoblotting with anti-FLAG or anti-GST antibody. Top images in each panel show anti-GST immunoblot (IB) of 4% of input. Middle and bottom images in each panel show blots for GST and FLAG, respectively, of the immunoprecipitate (IB). MW is shown on the right-hand side. (b) Co-IPs with putative interaction motif deletion constructs. Blot images on each side of respective rows were from the same membrane and were processed identically for brightness/contrast. (c) Co-IP with carboxy-terminal Hmx3a construct. FLAG antibody heavy and light chains appear at just above 50 kDa and at 25 kDa, respectively in IP:FLAG;IB:FLAG fractions. Note that though it lacks 9 amino acids vs full-length Hmx3a, FLAG-Hmx3a-Δeh1A runs slightly above full-length Hmx3a (panel (b) IP:FLAG;IB:FLAG). A non-specific band, likely corresponding to the yolk protein Vitellogenin, is also visible above GST-Tle3b-WDR in the anti-GST input fraction of Δeh1A and, very slightly, in the control in panel (b). n = at least 2 for all experiments. Original blots are presented in Supplementary Fig. S3.