Determination of EZH2 protein expression in endometrial carcinoma RL-952 cells by western blot analysis

After transfection, the total protein in the cells was extracted according to the RAPI lysis and extraction buffer manual. Protein concentration was quantified by Coomassie brilliant blue method. A total of 60 µg of protein was separated by 10% SDS-PAGE and the separated protein was transferred to PVDF membrane after 1.5 h of electrophoresis. The membrane was incubated in 5% skim milk powder at room temperature for 1 h, and then rabbit anti-human EZH2 primary antibody (1:1,500) was added and the membrane was incubated overnight at 4°C. After washing the membrane with PBS, the secondary antibody IgG (1:2,000) was added and the membrane was incubated again at 37°C for 2 h. ECL was added on the membrane and blots were developed in the dark. Images were recorded with a gel imaging system (Bio-Rad Laboratories, Irvine, CA, USA) and the gray-scale values were calculated. GADPH was used as the internal reference and the ratio of EZH2 to GAPDH protein was interpreted as the relative expression level of EZH2.