Western Blot Analysis of Cell Signaling

The si-NC and si-CHD5 siRNA were transfected into U87 cells with Lipofectamine 2000 reagent. After the cells were transfected for 48 h, Western blot analysis was performed as previously described [53 (link)]. Briefly, the cells were lysed with 1% SDS lysing buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Apexbio, Houston, TX, USA). The protein concentration was determined using the BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All the blots were incubated with the respective primary antibodies, namely, anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China), anti-CDK4, anti-CDK6, anti-CDK9 (1:800, Cell Signaling Technology, Boston, MA, USA), anti-E-cadherin, anti-N-cadherin, and anti-Twist1 (1:1000, Bioworld, Nanjing, China) antibodies. The protein bands were visualized with ECL Reagents (Smart-Lifesciences, Nanjing, China).