Smad2 3 fl 425

For western blot analysis cells were lysed using RIPA buffer containing PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Protein concentration was determined by Bradford assay (Bio‐Rad). Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences). The following antibodies were used: E2F1 (KH‐95; 1:500), EGFR (1:250), FGFR1 (Flg C15; 1:1000), TGFBR1 (V22; 1:1000), TGFBR2 (L21; 1:1000), Vimentin (V9; 1:1500), ZEB1 (H‐102; 1:1000), Snail (H-130; 1:500), SMAD2/3 (FL-425; 1:1000), and c-SRC (1:500) from Santa Cruz, E‐Cadherin (1:1500), and NFKB1 (C22B4; 1:1500) from Cell Signaling; N-Cadherin (610921; 1:1500) and FN1 (1:1000) from BD Bioscience, Actin (Sigma; 1:4000) and their corresponding HRP‐conjugated secondary antibodies (Pierce; 1:2000). Detection of HRP activity was performed with the ChemiDoc TouchTM Imaging System (BioRad) using ECL Plus (Amersham) or Super Signal West Femto (Thermo Scientific) Western Blotting Detection Reagents (GE Healthcare). Uncropped pictures of the immunoblots are shown in Supplementary Figs 9, 10, and 11.

Whole-cell extracts from H460 cells were prepared using cell lysis buffer containing 25 mM HEPES pH 7.5, 0.3 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 20 mM β-glycerophosphate, 0.1 mM Na₃VO₄, 0.1% Triton X-100 and complete protease inhibitor cocktail (P8340 Sigma, Madrid, Spain). Lysates were centrifuged and the supernatants transferred to a new tube. After adding SDS loading buffer the lysate was boiled for 5 min. 20 µg of protein extract was separated on SDS-polyacrylamide gel and electroblotted to Immobilon P membranes (Millipore, Burlington, VT, USA). Antibodies used were anti-p-ERK5 (3371, Cell Signaling, Danvers, MA, USA), anti-ERK5 (3372, Cell Signaling), anti-p-ERK1/2 (9106, Cell Signaling), anti-fibronectin (sc-71113, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p120 (P17920, BD Biosciences, Franklin Lakes, NJ, USA), γ-catenin (610254, BD Transduction, Franklin Lakes, NJ, USA), anti-β-tubulin (T9026, Sigma, St. Louis, MO, USA), phospho-Smad2/3 (Ser 423/425) and Smad2/3 (FL-425) were from Santa Cruz Biotechnology (Dallas, TX, USA). The signal was detected using enhanced chemiluminiscent (ECL) method (Santa Cruz Biotechnology). After washes the blots were re-incubated with the α-tubulin antibody to normalize for protein load.