Anti acly

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).

For western blot analysis, cells were washed with ice cold PBS and lysed in 1 x Cell Signaling lysis buffer (20 mM Tris-HCl, [pH 7.5], 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β- glycerophosphate, 1 mM Na₃VO₄, 1 μg/mL leupeptin (Cell Signaling Technologies), supplemented with 1 mM PMSF. Samples were frozen and thawed 3 times followed by centrifugation at 20,000 x g for 10 min at 4°C. Cleared protein lysate was denatured with LDS loading buffer for 10 min at 70°C, and loaded on precast 4% to 12% bis-tris protein gels (Life Technologies). Proteins were transferred onto nitrocellulose membranes using the iBLOT 2 system (Life Technologies) following the manufacturer’s protocols. Membranes were blocked with 5% w/v milk and 0.1% Tween-20 in TBS and incubated with the appropriate antibodies in 5% w/v BSA in TBS with 0.1% Tween-20 overnight at 4°C. All primary antibody incubations were followed by incubation with secondary HRP-conjugated antibody (Pierce) in 5% milk and 0.1% Tween-20 in TBS and visualized using SuperSignal West Pico or femto Chemiluminescent Substrate (Pierce) on Biomax MR film (Kodak). Antibodies used: anti-ODC, anti-DHPS, anti-DOHH, anti-β-Actin, anti-KAT3B (P300), α-tubulin (Abcam), anti-GAPDH, anti-KAT2A (GCN5), anti-ACLY (Cell Signaling), ant-EIF5A (BD Bioscience), anti-hypusine (Millipore).

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).

Total cellular protein was extracted using RIPA buffer (50 mM Tris-HCl pH 8.0 containing 1% NP-40, 150 mM NaCl, 5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride) containing protease and phosphatase inhibitors. The total protein was quantified using Bradford reagent. Equal amounts of protein lysates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were blocked with 5% skimmed milk for 2 h at room temperature. The blots were incubated with different primary antibodies overnight at 4 °C, followed by the appropriate horseradish peroxidase-conjugated secondary antibody (1:5,000) for protein visualization with ECL reagents (Millipore). We utilized the following primary antibodies: anti-ACLY (Cell Signaling, 4332), anti-ACeCS1 (Cell Signaling, 3658), anti-SAT1 (Cell Signaling, 61586), anti-SP1 (Cell Signaling, 9389), anti-acetyl-lysine (Cell Signaling, 9441), anti-β-actin (DHSB, JLA20), anti-acetyl-histone H3 (Lys9) (Cell Signaling, 9649), anti-acetyl-histone H3 (Lys27) (Cell Signaling, 4353), anti-acetyl-histone H3 (Lys18) (Cell Signaling, 9675), anti-histone H3 (Abcam, ab1791), anti-ACLY (Abcam, ab40793), anti-H3K27 (Active Motif, 39133) and HA antibody (Covance, MMS-101R). Protein expression levels were normalized to the respective loading control, β-actin.