Anti phospho smad2

Blastula explants (20 explants/sample) were collected for specified stage / lineage and lysed in TNE phospho-lysis buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCL, 0.5 mM EDTA, and 0.5% Triton X-100, 2 mM Sodium Orthovanadate, 20 mM Sodium Fluoride, 10 mM B-Glycerophosphate, 1 MM Sodium Molybdate dihydrate] supplemented with protease inhibitors [Aprotinin, Leupeptin and phenylmethylsulfonyl fluoride (PMSF)] and a PhosStop phosphatase inhibitor and a complete Mini tablet (Roche). SDS-PAGE and western blot analyses were used to detect proteins and modifications using the following antibodies: anti-phospho SMAD2 (Ser465/467, Sigma, 1:500), Smad2 Polyclonal Antibody (Life Technologies 1:500) Anti-phospho Smad1/Smad5/Smad8 (Ser463/465, Sigma, 1:1000), Smad1 (D59D7, Cell Signal, 1:1000), Actin (A2066, Sigma-Aldrich, 1:5000). For chemiluminescence-based detection, horseradish peroxidase (HRP)-conjugated rabbit secondary antibodies were used (Vector Laboratories, 1:20,000). Blots were cut to the appropriate size based on the protein standard ladder before hybridization with antibodies in order to conserve antibody. Results shown are representative of at least three independent experiments. Raw images of blots in the main figures are included as Supplemental Fig. 8 and raw images of blots in supplemental figures are included as Supplemental Fig. 9.

Total protein extracts were generated using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with PhosSTOP and Complete Phosphatase/Protease Inhibitor Cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Protein extracts (20–25 μg per sample) were loaded onto SDS-PAGE gels and transferred electrophoretically to PVDF membranes and immunodetection of proteins was performed using ECL™ Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK). The following primary antibodies were used: anti-Myc, anti-HIF1a, anti-4EBP1, anti-eIF4E, anti-YBX1, anti-Snail, anti-CA9, anti-Smad2/3, anti-β-catenin (Cell Signaling), anti-phospho Smad2 (Millipore), anti-β3 integrin, anti-MMP3, anti-N-cadherin (Abcam), anti-CycD1, anti-vimentin (Santa Cruz Biotechnology), anti-BMP2, anti-CCDC103, anti-TTC30B, anti-EIF3G, anti-RPL11 (CusaBio), anti-Cx31 (Alpha Diagnostics), anti-eIF4E2 (GeneTex), anti-αv integrin and anti-β-actin (1:500; Calbiochem, Darmstadt, Germany). Anti-mouse and anti-rabbit HRP secondary antibodies were from Pierce. Bound antibodies were visualized with an enhanced chemiluminescence detection kit (Amersham Pharma-Biotech).