Ezh2 antibody

ChIP assays were carried out with EZ-CHIP Kit following the manufacturer’s recommendations (Millipore, Billerica, MA, USA). EZH2 antibody was purchased from Millipore (Billerica, MA, USA) and H3 trimethyl Lys 27(H3K27me3) antibody was from Abcam (Cambridge, UK). The sequences of ChIP primer are shown in Supplementary Table S1. We calculated the ChIP data as a proportion of the added DNA using the equation ₂[input Ct − target Ct] _{× 0.1 × 100}.

Total protein extracts were obtained by lying cells in ice-cold lysis buffer. The proteins were separated on SDS-polyacrylamide gels and transferred to PVDF membranes (Bio-Rad). After blocking in 5% skimmed milk for 1 h, the membranes were incubated with a primary antibody overnight at 4°C. Membranes were washed 3 times for 10 min in TBST and incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. After washing 3 times for 10 min in TBST, the proteins were visualized with an ECL detection system. The EZH2 antibody was purchased from Millipore, the ERα antibody was from Santa Cruz, and the β-actin antibody was obtained from Sigma-Aldrich.

Chromatin immunoprecipitation (ChIP) assays were performed using the EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore) and EZH2 antibody (Millipore) or H3K27me3 antibody (Millipore) following the manufacturer’s protocol. Retrieved DNA was measured using SYBR® Premix Ex Taq™ II (Takara) on ABI StepOnePlus system (Applied Biosystems). The primer sequences were as follows: 5′-CTGCGTCACCGTCACTGG-3′ (forward) and 5′-ACAACTCGCCCGTCTCTG-3′ (reverse) for miR-200b/a/429 promoter; 5′-GCTGGGCGTGACTGTTAC-3′ (forward) and 5′-GAGTGTGGTGTTGGGGGA-3′ (reverse) for β-actin promoter.

RIP assays were conducted using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) in accordance with the manufacturer’s protocols. The EZH2 antibody for RIP assays was obtained from Millipore (Billerica, MA, USA).