Figure 1. TGFβ and BMP induce Smad C-tail and linker phosphorylation.

(A) Nuclear and cytoplasmic fractions of BMP or TGFβ treated (1h) HaCaT cells were analyzed by western blot with the indicated antibodies. (B) and (C) HaCaT cells treated with BMP, TGFβ or no addition (control) for 1 h were fixed and analyzed by immunofluorescence (IF) with antibodies against Smad1 (pLinker, Smad1 pS206; pTail, Smad1/5 pTail) and Smad3 (pLinker, Smad3 pS213; pTail, Smad3 pTail) including DAPI-staining of nuclei. (D) As in (B), with BMP treatment for the indicated times. (E–I) Images of E13.5 mouse embryo sections. (E) Adjacent sections through the telencephalic ventricular zone immunohistochemically stained with anti-Smad1 antibodies described in (B and C), with Hematoxylin counterstaining. Images at 40x magnification. Ventricle and ventricular zone (VZ) are indicated. (F) Confocal images of a section similar to (A) with double-immunofluorescence staining using the indicated antibodies and DAPI counterstaining. Images are at 20x magnification, with the far right panel at 200x. The ventricular zone is indicated by white dashed lines. (G) Immunohistochemistry on adjacent sections through dorsal root ganglia stained as in (E), with antibodies against Smad2 (pLinker Smad2, Smad2 pS245/250/245; pTail Smad2). Images at 80x magnification. (H) Confocal images of dorsal root ganglia with double-immunofluorescence staining with the antibodies used in (G). Magnification was 80x. (I) Merged confocal image of E13.5 testis from the same section as in (H).