Anti pparα

Total protein of each sample was prepared and quantified by the bicinchoninic acid method (Pierce, Rockford, USA). After their electrophoretical separation on 12% SDS-PAGE gels, these protein samples were transferred onto polyvinylidine difluoride membranes and blocked by 5% nonfat dry milk (NFDM). Membranes with protein sample subsequently reacted to anti-PPARα (HepG2, 1 : 500, Santa Cruz, USA; liver: 1 : 1000, Santa Cruz, USA), anti-CPT2 (HepG2, 1 : 1000, Santa Cruz, USA), anti-ACBD3 (HepG2, 1 : 1000, Santa Cruz, USA), anti-GAPDH (HepG2, 1 : 1000, Santa Cruz, USA), and β-actin (liver, 1 : 200, Boster, China) overnight at 4°C and then HRP-conjugated secondary antibody (1 : 1500; Jackson ImmunoResearch Laboratories, Inc., USA) for 1 hour at room temperature. Both chemiluminescent visualization by ECL detection system and densitometric analysis by Image Lab Software 5.1 (Bio-Rad Laboratories, USA) were carried out to assess the immune signals specific to immunoblots [33 (link)].

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.

Dexamethasone (D4902) (DEX) and GW7647 (G6793) (GW) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Anti-GR, anti-PPARα, anti-RNA pol II and anti-p65 antibodies were obtained from Santa Cruz. Phospho-specific rabbit antibodies to p38 (Thr-180/Tyr-182), p42/44 ERK (Thr202/Tyr204), MSK1 (Thr581) and IKKα/β (Ser180/S181) were used to detect the respective phosphorylated forms and purchased from Cell Signaling. Anti-p38, anti-ERK, anti-MSK1, and anti-IκBα antibodies were purchased from Cell Signaling. Anti-tubulin and anti-actin were used as loading control and obtained from Santa Cruz. Anti-phospho-65 was obtained from Santa Cruz. Recombinant murine TNFα was produced and purified as described (30 (link)). TNFα was used at a final concentration of 2,000 IU/ml. p(IL6κB)₃50hu.IL6P-luc+ (hereafter renamed NF-κB-Luc), PPARα, GR, and 5HT7 control plasmids were described previously (21 (link), 31 (link)–33 (link)). LPS was purchased from Invivogen.