Anti p acc

The protein expression in the liver was determined through Western blotting following a previously described protocol [20 (link)]. The membranes were then incubated with primary antibody (PPARγ, IL-6, IL-1β, SIRT1, LXRα, pAMPKα, AMPKα, PPARα, pACC, ACC, FAS, SREBP1c, CPT1B, and β-actin) at room temperature for 2 h. In this study, the primary antibodies were anti-PPARγ (Cat# 2435S, 1:1000, Cell Signaling, Danvers, MA, USA), anti-IL-6 (Cat# 21865-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-IL-1β (Cat# 16806-1-AP, 1:1000, Proteintech), anti-SIRT1 (Cat# 9475S, 1:1000, Cell Signaling), anti-LXRα (Cat# ab176323, 1:1000, Abcam, Cambridge, UK), anti-pAMPKα (Cat# AF3423, 1:1000, Affinity, San Francisco, CA, USA), anti-AMPKα (Cat# AF6423, 1:1000, Affinity), anti-PPARα (Cat# sc-398394, 1:500, Santa Cruz, Santa Cruz, CA, USA), anti-pACC (Cat# D7D11,1:2000, Cell Signaling), anti-ACC (Cat# C83B10, 1:2000, Cell Signaling), anti-FAS (Cat# C20G5, 1:2000, Cell Signaling), anti-SREBP1c (Cat# ab28481, 1:2000, Abcam), anti-CPT1B(Cat# DF3904, 1:500, Affinity), and anti-β-actin (Cat# GTX109639, 1:10000, Genentech, San Francisco, CA, USA). The relative expression of proteins was quantified densitometrically using ImageJ software (Wayne Rasband, Madison, WI, USA), and β-actin was used as the internal control.

A-769662 was obtained from Abcam (Cambridge, MA), AICAR was obtained from Cell Signaling Technology (Danvers, MA). Sulfo-NHS-SS-biotin was obtained from Pierce (Thermo Fisher Scientific, Rockford, IL). Antibodies used for immunoblotting were as follows: anti-EGFR from Genetex (Irvine, CA), anti-CHC from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pACC, anti-AMPK (α1/2), anti- actin, and anti-Erk from Cell Signaling Technology (Danvers, MA), and anti-ZNF142 antibodies from Aviva Systems Biology (San Diego, CA). Antibodies used for immunofluorescence microscopy were as follows: anti-β1-integrin from EMD Millipore, Darmstadt, Germany), and anti-TfR from Santa Cruz Biotechnology (Santa Cruz, CA).

Mouse liver homogenates were prepared using a RIPA buffer containing protease and tyrosine phosphatase inhibitor cocktail (Sigma). The protein concentration of the lysates was determined using the Bicinchoninic (BCA) Protein Assay Kit (Thermo Scientific, IL, USA), according to the manufacturer’s instructions. The isolated soluble proteins (20 mg) were separated on 8–15% SDS-polyacrylamide gels. The separated proteins were then electroblotted onto nitrocellulose transfer membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with 5% skim milk for 1 h and then probed with the following 1:1,000-diluted antibodies: anti-pAMPKa, anti-AMPKa, anti-pACC, anti-ACC, anti-CYP2E1, anti-CPT1a (Cell Signaling Technology), and anti-β-actin (Santa Cruz Biotechnology) for 18 h at 4°C. After 3 washes for 10 min each, polyclonal anti-rabbit or mouse HRP-conjugated secondary antibodies (Santa Cruz Biotechnology), which were linked to HRP-bound protein complexes and developed with a Pierce ECL Western Blot substrate (Thermo Fisher Scientific), were added to the mixture. Band densitometry was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA) and expressed as fold change relative to β-actin.

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).