Rabbit anti atm

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.

Cells harvested, pelleted at 1,500 rpm at 4°C for 5 min and lysed with whole cell lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, and cocktail protease inhibitors). Protein concentration was determined using the Bradford assay (Sigma). Protein bands were separated on a NuPAGE 4–12% Bis-Tris Gel (Invitrogen). Proteins were electrotransferred onto a nitrocellulose membrane in 1X NuPAGE transfer buffer. After blocking with 5% skimmed milk solution in 1% Tris-Buffered saline with Tween 20 (TBST) buffer, the membranes were immunoblotted with mouse anti-Flag antibody (Sigma, #F3165, 1:1000), rabbit anti-TDP-43 (Proteintech, #10782-2-AP, 1:1000), mouse anti-phospho-Histone H2.AX (S139) (EMD Millipore, #16-193, 1:1000), rabbit anti-H2.AX (Cell Signaling Tech, #2595, 1:1000), rabbit anti-phospho-ATM (S1981) (Abcam, #ab81292, 1:800), rabbit anti-ATM (Abcam, #ab32420, 1:1000), rabbit anti-phospho-53BP1 (S1778) (Cell Signaling Tech, #2675, 1:1000), rabbit anti-53BP1 (Cell Signaling Tech, #4937, 1:1000), and mouse anti-β-actin (Proteintech, #66009-1-Ig, 1:5000) antibodies. Protein bands were visualized by probing with corresponding HRP-conjugated secondary antibodies and developed with enhanced chemiluminescence reagent in Odyssey (LI-COR).