Ecl reagents

The Flag-CTL and Flag-GDPD5 plasmids were transfected into SH-SY5Y cells with Neofect™ DNA transfection reagent. After cells were transfected for 48 h, the cells were lysed by 1% SDS lysing buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Apexbio, Houston, TX, USA) for Western blot analysis. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All blots were, respectively, incubated with primary anti-bodies anti-flag (1:1000, ABclonal, Wuhan, China), anti-p-ACC, anti-ACC, Anti-p-ACLY, anti-ACLY, anti-PFKFB3, anti-HK2 and anti-LDHA (1:1000, Cell Signaling Technology, MA, USA), anti-ALDOA, anti-ENO2, anti-HADH and anti-PPARA (1:1000, proteintech, Wuhan, China), as well as anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China). Bands were visualized with ECL Reagents (Smart-Lifesciences, Changzhou, China).

The GFP-CTL and GFP-HMGCS2 plasmids were transfected into OSRC2 cells with Neofect™ DNA transfection reagent. After cells were transfected for 48 h, the cells were lysed by 1% SDS lysing buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Apexbio, Houston, TX, USA) for Western blot analysis. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All blots were, respectively, incubated with primary anti-bodies anti-flag (1:1000, ABclonal, Wuhan, China), anti-p-ACC, anti-ACC, Anti-p-ACLY, anti-ACLY, anti-PFKFB3, anti-HK2 and anti-LDHA (1:1000, Cell Signaling Technology, MA, USA), anti-ALDOA, anti-ENO2, anti-HADH and anti-PPARA (1:1000, proteintech, Wuhan, China), and anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China). Bands were visualized with ECL Reagents (Smart-Lifesciences, Changzhou, China).

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).