Rabbit anti phospho smad1 5 8

Immunostaining was done using standard whole mount immunostaining protocol. Fixed embryos were washed three times 30 min in PBSTx (PBS containing 1% [v/v] TritonX‐100) and blocked with 10% (v/v) goat serum/PBSTx/2% (w/v) NaN3 for 90 min, followed by an overnight incubation at room temperature in primary antibody, rabbit anti‐phospho‐SMAD1/5/8 at 1:300 (Cell Signalling #13820). Embryos were washed three times in block and then incubated overnight at room temperature in an appropriate Alexa conjugated secondary antibody (Molecular Probes; 1:1000). Embryos were then washed in PBSTx, bisected or sectioned and photographed using a fluorescence microscope.

Immunoblotting was performed according to standard procedures. In brief, a piece of pancreas was frozen in liquid nitrogen immediately after killing the mice. Protein lysates were prepared using protein extraction buffer (4% SDS, 100 mM Tris-HCl) containing protease inhibitors and 1 mM phenylmethylsulfonyl fluoride (PMSF), and cell debris was removed by centrifugation at 4 °C for 10 min at 14,000 r.p.m. Protein content was measured using a colorimetric assay (Bradford Biorad Assay, Biorad). Lysates (30 μg) were resolved by SDS–PAGE and transferred to a PVDF membrane (#IPVH00010, Immobilon-P Membrane, Millipore). Immunoreactive bands were visualized using chemiluminescence (Thermo scientific, Waltham, MA, USA) (Supplementary Fig. 6). Images were processed and analysed using the ImageJ software (http://rsbweb.nih.gov/ij/). Antibodies used are as follows: goat anti-Bmp4 (#sc-6896 Santa Cruz); rabbit anti-Nodal (#39953, Abcam) rabbit anti-Phospho Smad 1/5/8 (#9511, Cell Signaling); rabbit anti-Phospho Smad 2/3 (#3101, Cell Signaling); rabbit anti-ATM (#ab78, Abcam) all 1:1000, O.N. at 4° and mouse anti-β-actin (#3101, Sigma) 1:50,000 for 1 h at RT.