Mouse anti human anti gapdh antibody

Following transfection, the cells were lysed with the radioimmunoprecipitation assay lysis buffer containing phenylmethane sulfonyl fluoride (Beyotime Institute of Biotechnology). Subsequent to centrifugation at 12,000 × g and 4°C for 10 min, the protein concentration was measured with the BCA method. A protein sample (5 µl) was then subjected to 12% SDS-PAGE and electronically transferred onto a polyvinylidene difluoride membrane. After blocking with non-fat milk at room temperature for 1 h, the membrane was incubated with rabbit anti-human anti-TIMP2 antibody (1:1,000 dilution; cat. no. ab180630; Abcam, Cambridge, UK) or mouse anti-human anti-GAPDH antibody (1:5,000 dilution; cat. no. ab8245; Abcam) at 4°C overnight. Subsequently, the membrane was incubated with a horseradish peroxidase-conjugated goat anti-mouse (1:5,000 dilution; cat. no. ab6789; Abcam) or goat anti-rabbit (1:2,000 dilution; cat. no. ab6721; Abcam) secondary antibodies, respectively, at room temperature for 1 h. Color development was performed with the enhanced chemiluminescence kit (Beyotime Institute of Biotechnology), and the protein bands were then imaged and analyzed using Quantity One software (version 4.62; Bio-Rad, Hercules, CA, USA).

When MDA-MB-231 cells reached 70–80% confluence, they were collected and lysed with RIPA (cat. no. P8340; Merck KGaA) at 72 h after EZH2 adenovirus infection, and total protein was extracted. Protein concentration was quantified with a BCA kit and then detected by western blotting. A total of 50 µg protein/lane was separated by 10% SDS-PAGE, transferred to PVDF membranes (cat. no. 03010040001; Roche) and blocked with 5% skimmed milk at 25°C for 1 h. Rabbit anti-human EZH2 polyclonal antibody (cat. no. ab186006; 1:1,000 dilution; Abcam) and mouse anti-human anti-GAPDH antibody (cat. no. D190090; 1:3,000 dilution; Sangon Biotech) were added and membranes were incubated overnight at 4°C. Subsequently, the PVDF membranes were washed three times with 20% Tween-20 in tris-buffered saline (TBST) and incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (1:5,000) for 2 h at 25°C. Next, the membrane was washed three times with TBST. Protein bands were visualized using ECL. Performing grayscale analysis of Western Blot bands using ImageJ (version: 1.54D; National Institutes of Health).