Rabbit anti human ezh2 antibody

Osteosarcoma cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) containing protease inhibitors (Sigma-Aldrich). Protein quantification was done using a BCA protein assay kit (Promega). The primary antibodies used for western blotting were rabbit anti-human EZH2 antibody (#4905S, 1:1000; Cell Signaling Technology), rabbit anti-human β-actin antibody (#4967S, 1:1000, Cell Signaling Technology), and rabbit anti-human E-cadherin antibody (#sc-21791, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (#sc-2004, 1:5000; Santa Cruz Biotechnology) were used as the secondary antibodies. A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). Then, the membrane was blocked with 5% (5 g/100 mL) nonfat dry milk (Bio-Rad, CA, USA) in tri-buffered saline plus Tween (TBS-T) buffer for 2 h. Blots were immunostained with primary antibody at 4 °C overnight and with secondary antibody at room temperature for 1 h. Immunoblots were visualized using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore) and calculated with Image J 1.47 software. Protein levels were normalized to β-actin.

LAD cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) containing protease inhibitors (Sigma-Aldrich). Protein quantification was done using a BCA protein assay kit (Promega). A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). Then, the membrane was blocked with 5% (5 g/100 mL) nonfat dry milk (Bio-Rad, CA, USA) in tri-buffered saline plus Tween (TBS-T) buffer for 2 h. Blots were immunostained with primary antibody at 4 °C overnight and with secondary antibody at room temperature for 1 h. Immunoblots were visualized using Immobilon^TM Western Chemiluminescent HRP Substrate (Millipore). Protein levels were normalized to β-actin. The primary antibodies used for western blotting were rabbit anti-human EZH2 antibody (#4905S; 1:1000; Cell Signaling Technology), rabbit anti-human BAX antibody (#2772; 1:1000, Cell Signaling Technology), and rabbit anti-human β-actin antibody (#sc-47778; 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (1:5000; Santa Cruz Biotechnology) were used as the secondary antibodies.