Ab157464

Cell extracts were prepared by incubating the cells in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5% Nonidet P-40 [NP40]) for 30 min at 4°C, followed by centrifugation at 13,000 rpm for 15 min at 4°C. The protein concentrations of the total cellular lysates were measured using the micro bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA). For immunoprecipitation, 500 μg of protein was incubated with specific antibodies (2–3 μg) for 12 h at 4°C with constant rotation, followed by incubation with 30 μL of 50% protein A/G agarose beads for 2 h. Beads were then washed five times with PBS. The precipitates were boiled in 2× SDS-PAGE loading buffer for 10 min, and then resolved in 10% SDS-PAGE gels. The proteins in the gel were transferred to polyvinylidene fluoride membranes. For western blotting analysis, membranes were incubated with antibodies, including MTA1 (1:1,000, #5646, Cell Signaling Technology), ZEB2 (1:1,000, ab138222, Abcam), E-cadherin (1:1,000, #14472, Cell Signaling Technology), vimentin (1:1,000, #5741, Cell Signaling Technology), MBD3 (1:1,000, ab157464, Abcam), and HDAC1 (1:1,000, ab19845, Abcam), overnight at 4°C followed by incubation with a secondary antibody. Immunoreactive bands were visualized using western blotting luminol reagent (Santa Cruz Biotechnology).