Fig. 2. Noggin1 and Noggin2 can bind TGFβ and Wnt ligands. (A) Selected mRNA and proteins used in the present study. (B,C) Comparison of translation capacities of MycNoggin1Δ5 and MycNoggin2Δ5 mRNA (B) or MycNoggin2Δ5, SynMycNoggin1 and SynMycNoggin2 mRNA (C) injected in two-cell embryos at the indicated concentrations. Five embryos of each type were collected at stage 10 in 50 μl of lysis buffer and Noggin proteins were revealed by western blotting with anti-Myc antibody either in 1/5 (B) or in 1/125 (C) aliquots of this volume. Here and below, α-tubulin was detected with anti-tubulin antibodies (DM1A, Sigma, final dilution 1:10,000) as a loading control. (D) qRT-PCR analysis of endogenous Noggin1 and Noggin2 mRNA in the anterior neural fold explants of stage 15 embryos. (E,F) Only endogenous Noggin2 (lane 2), but not Noggin1 (lane 1), was detected in the anterior neural fold explants of stage 15 embryos by antibodies specific to Noggin1 and to Noggin2 (E), despite these antibodies demonstrating similar affinities to exogenous Noggin1 (lane 3) and Noggin2 (lane 4) translated from injected SynNoggin1 and SynNoggin2 mRNA (F). In the last case, a mixture of antibodies to both Noggin proteins was used. (G,H) Both Noggin1 and Noggin2 (Ng1 and Ng2) translated from SynMycNoggin1 and SynMycNoggin2 mRNA co-precipitate with Flag-tagged BMP4, ADMP, Activin, Xnr2, Xnr4 and XWnt8. In case of Noggin1 translated from MycNoggin1Δ5mRNA (wtNg1), only precipitation with BMP was detected. No precipitation of Noggin proteins was revealed with Flag-tagged Zyxin (negative control). (I) Deletion of the clip-domain sharply reduce ability of Noggin proteins (ΔNg1, ΔNg2) to bind BMP4 but much more poorly influences the binding of Noggin to all non-BMP TGFβ ligands tested and to XWnt8.
[2.8.5]
