Zebrafish Prmt2, Azin1b, Hmgn3, Hmgb1a, and the WDR domain of Tle3b bind full-length zebrafish Hmx3a in Co-IPs and have varying interaction profiles with proteins encoded by hmx3a mutant alleles. (a) Schematics of FLAG-tagged Hmx3a constructs used in these experiments. SU42, sa23054, SU43, and SU3 indicate different mutant forms of Hmx3a (See 5). eh1A, eh1B, and WYPY indicate predicted interaction motifs for Tle3b-WDR. Numbers indicate residues encompassing annotated domains or the total number of WT residues in each protein. (b–f) Putative binding partners were expressed as GST-fusion proteins, purified, and incubated with either a FLAG-Hmx3a-expressing embryonic 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 (IP). The Hmx3a protein included in each lysate is indicated along the top x axis. Molecular weight (MW) is shown on the right-hand side. Prey proteins were (b) Prmt2, (c) Azin1b, (d) Hmgn3, (e) Hmgb1a, and (f) the WDR domain of Tle3b. FLAG antibody heavy and light chains appear at just above 50 and at 25 kDa, respectively in IP:FLAG;IB:FLAG fractions. Non-specific signal from the FLAG antibody heavy chain is also slightly visible in IP:FLAG;IB:GST fractions in (d) and (e). In all cases, the highest MW band in anti-GST input fractions is the full-length GST fusion protein. n = at least 2 for all experiments. Original blots are presented in Supplementary Fig. S3.
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