Protease inhibitor cocktail and phosphatase inhibitor cocktail

Protein was extracted from cells using RIPA lysis buffer (Beyotime, China) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (APExBIO, Houston, USA) and its concentration was detected with BCA assay (Beyotime, China). 20 μg of total protein from each sample or PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA) was diluted in loading buffer (Beyotime, China) for immunoblot. Proteins were separated with SDS-PAGE and transferred to a nitrocellulose blotting membrane. Membranes were blocked in 5% BSA for 1.5 h, and incubated with primary antibodies overnight. Then membranes were washed by PBST and continued to be incubated with HRP-conjugated secondary antibodies for 1 h. The protein bands were visualized by ECL Enhanced Kit (ABclonal, China) and detected by Bio-Rad ChemiDoc™ Touch Imaging System. Antibodies against p21, Cyclin D1, PUMA, Bcl-2, Bax, Caspase-9, NF-κB p65 and Phospho-NF-κB p65-S536 were used at a dilution of 1: 1,000. β-Actin and secondary antibodies were used at a dilution of 1: 8,000.

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).