Fig. 3

a WT HaCaT cells and two clones of SMAD4 knockout (KO) HaCaT cells were treated or not with BMP4. DNA pulldowns from whole cell extracts were performed using an oligonucleotide containing the SMAD1/5–SMAD4 binding sites of the upstream ID3 enhancer, or a version in which these sites were mutated. Pulldowns were immunoblotted with the indicated antibodies and inputs are shown below. The immunoblot is representative of three independent experiments. Molecular weight markers are given in kDa on the right of the blots. b Immunostaining for pSmad1/5 (green) in 40% epiboly CTRL and MZsmad4a embryos. Nuclei are stained with DAPI (blue). The bottom panel highlights the pSmad1/5 gradient without the DAPI channel. Scale bars correspond to 100 µm. Animal views are shown. c Quantitation of the pSmad1/5 gradient size with respect to the embryo surface. 14 embryos for each group are represented as means ± SD. p = 4.985 × 10^-8. Two sided Mann–Whitney test. ****p < 0.0001. d As in b but showing CTRL and MZsmad4a embryos injected with 60 pg of hBMP4 mRNA. Scale bars correspond to 100 µm. e Quantitation of hBMP4 mRNA-injected CTRL and MZsmad4a embryos. Six embryos for each group are represented as means ± SD. Two sided Mann–Whitney test. ns not significant.