H3k27me3

786-0 and ACHN cells were washed with PBS buffer twice and then homogenized in 200μl radioimmuno-precipitation assay (RIPA) buffer containing the protease inhibitors cocktail(1 mmol/L) and phenylmethylsulfonyl fluoride (100μg/mL). Homogenates were centrifuged and supernatants were collected. A total of 50 μg of protein separated by 10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane were saturated with 5% skim milk in TBST (50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20) for 2h and then incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study included rabbit polyclonal antibodies to UTX (1:1,000, Abcam, Hong Kong, China), JMJD3 (1:1500, Abcam), EZH2 (1:500, Santa Cruz Biotechnology, Hong Kong, China), H3K27me3 (1:1,500, Epigentek, Brooklyn, USA), H3 (1:2,000, Sigma-Aldrich, St Louis, USA) and actin (1:2,500, Sigma, St Louis, USA). The membranes were incubated with HRP-conjugated goat anti-rabbit antibody (1:5,000, Sigma) for 1 h at room temperature and then exposed to enhanced chemiluminescence substrate (Millipore, Rockford, USA), and detection was performed using a film.

The tissues and cells were added radioimmuno-precipitation assay(RIPA) buffer containing the protease inhibitors cocktail (1 mmol/L) and phenylmethylsulfonyl fluoride (100μg/mL), and sonicated to prepare homogenates. Homogenates were centrifuged and supernatants were collected. 50 μg protein was separated by 10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were saturated with 5% skim milk in TBST for 2h and then incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study included rabbit polyclonal antibodies to JMJD3 (1:1500, Abcam, Hong Kong, China), H3K27me3 (1:1,500, Epigentek, Brooklyn, USA) and β-Actin (1:2,500, Sigma, St Louis, USA). The membranes were incubated with HRP-conjugated goat anti-rabbit antibody (1:5,000, Sigma) for 1 h at room temperature and then exposed to enhanced chemiluminescence substrate (Millipore, Rockford, USA), and detection was performed using a film.