Genegnome detection system

Equal amounts of total protein per sample were diluted with NuPAGE® LDS Sample Buffer (Novex, Life Technologies, Carlsbad, CA) and reduced with dithiothreitol (DTT). Electrophoresis was performed with NuPAGE® Novex® 4–12% Bis-Tris Gels (Novex, Life Technologies, Carlsbad, CA) and proteins were transferred to Hybond-C Extra nitrocellulose membrane (Amersham Biosciences, GE Healthcare, Piscataway, NJ). The following concentrations of primary antibody were used: TGFβ2 (1:200), phospho-ATF-2 (1:200), ATF-2 (1:200), phospho-Smad2 (1:1000), Smad2 (1:1000), β-actin (1:7000). Following primary antibody incubation, membranes were incubated in appropriate secondary antibodies and images were developed in Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA) prior to visualization using the GeneGnome detection system (Syngene, Frederick, MD).

HCT116 cells (1.5 × 10⁶ cells) were seeded into 10-cm cell culture dishes and incubated for 24 h. Then cells were treated with DZNep (5 µM) for 1, 3, 6, 24, and 48 h. Untreated HCT116 cells were served as control. After the indicated incubation periods, cells were harvested, washed in PBS, and proteins were extracted by lysis with Urea Lysis Buffer. Protein concentration was measured using the Bio-Rad DC™ Protein Assay (BioRad). Equal amounts of lysates were applied to SDS-PAGE and proteins were then transferred to a nitrocellulose membrane (Amersham Protran Premium 0.2 NC, GE Healthcare Life Sciences) prior to probing with antibodies. Antibodies were applied as follows: anti-EZH2 (1:10,000, EZH2 (D2C9) XP, monoclonal rabbit, Cell Signaling), anti-H3K27me3 (1:10,000, Tri-Methyl-Histone H3 (Lys27) (C36B11, monoclonal rabbit, Cell Signaling), anti-GAPDH-HRP as loading control (1:50,000, clone 6C5, monoclonal mouse, Abnova), and secondary HRP-conjugated antibody (1:10000, Goat anti-Rabbit IgG (H + L, Thermo Scientific). Signal of protein bands was detected using chemiluminescent HRP substrate (Immobilon™ Western Chemiluminescent HRP substrate, Millipore) according to the manufacturer’s instructions and the GeneGnome detection system (Syngene).