Chemocam

Proteins were loaded in equal amounts and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck Millipore). Membranes were incubated with the primary antibodies directed against the following molecules: α-SMA (Sigma-Aldrich) PDGF receptor-β (Cell Signaling Technology, Danvers, MA), collagen 1α1 (R&D Systems, Minneapolis, MN), JNK, phospho-JNK (pJNK), ERK1/2, phospho-ERK1/2 (pERK), SMAD2, phospho-SMAD2 (pSMAD2), AKT, phospho-AKT (pAKT), p70S6K, phospho-p70S6K (pp70S6K) (all from Cell Signaling Technology), GAPDH (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich). The following secondary antibodies were used as needed: a goat-anti-mouse-IgG-HRP antibody (Bio-Rad, Feldkirchen, Germany), a goat-anti-rabbit-IgG-HRP (Bio-Rad), or a goat-anti-sheep-IgG-HRP antibody (R&D Systems). Visualization was performed using the ChemoCam (INTAS, Homburg, Germany) after incubation with Clarity Western ECL Substrate (Bio-Rad).

The treated and non-treated control cells were harvested after 48 h and lysed in ice-cold RIPA lysis buffer (50 mM Tris; 250 mM NaCl; 2 % Nonidet-P40; 2.5 mM EDTA; 0.1 % SDS; 0.5 % DOC; complete protease inhibitor; 1.0 % phosphatase inhibitor; pH 7.2). Protein concentration was determined by micro-Lowry. Thirty micrograms of total protein was separated by SDS-PAGE and transferred to nitrocellulose membranes (Roth, Karlsruhe, Germany). The membranes were blocked by 5 % blocking buffer (milk powder in Tris-buffered saline Tween (TBST)) for 1 h and incubated overnight with Snail, GADD48B, P21, cyclin B1, E-cadherin, TET2, TET3, and GAPDH mouse/rabbit polyclonal primary antibodies at 4 °C. The following day, the membranes were incubated with the corresponding HRP-labeled secondary antibodies for 1 h at RT. Chemiluminescent signals were detected with the ChemoCam (INTAS, Göttingen, Germany).

Protein samples were separated using 10% SDS polyacrylamide gels and transferred to nitrocellulose, membranes were blocked with 5% dry milk in TBST (0.1% Tween20 in TBS). Primary antibodies were diluted in blocking solution: anti-Myc (1:2500, Santa Cruz Biotechnology, sc-40), anti-His (1:2500, Thermo Fisher Scientific, MA1-21315), anti-GST (1:20,000, Cell Signaling Technology, 26H1), anti-pTyr (1:1000, Santa Cruz Biotechnology, sc-508). Protein bands were detected with a ChemoCam (Intas) using HRP-coupled secondary antibodies (GE Healthcare) and ECL Prime Western Blotting Detection Reagent (GE Healthcare).