Srebp2

Protein isolation from both tissue and cells were done as mentioned earlier. Primary antibodies against DJ-1, LC3B (Abcam, MA, USA), SREBP-2 (Santa Cruz, CA, USA), CHPV P22 (link) at 1:1000 and LDLR (Abcam, MA, USA) at 1: 500 dilutions, were used for studying the expression levels of respective proteins. The blots were processed for development using chemiluminescence reagent (Millipore, MA, USA). The images were captured and analyzed using Chemigenius Bioimaging System (Syngene, Cambridge, UK). To determine equivalent loading of samples the blots were stripped and reprobed with β-Actin (Sigma Aldrich, USA) (whole cell), HSP60 (Abcam, MA USA) (mitochondria), PCNA (Cell Signalling, MA, USA) (nuclear).

Differentiated THP-1 cells were transfected with 30 nM small interfering RNA (siRNA) for LXRα (Santa Cruz, Dallas, TX, USA, #sc- 38828), LXRβ (Santa Cruz, #sc-45316), SREBP1 (Santa Cruz, Dallas, TX, USA, #sc-36557), SREBP2 (Santa Cruz, Dallas, TX, USA, #sc-36559) or Scramble RNA (Santa Cruz, Dallas, TX, USA, #sc-37007) using the Lipofectamine RNAiMAX Transfection Reagent according to the manufacturer’s instructions (Thermofisher, Carlsbad, CA, USA, #13778075). The knockdown efficiency was confirmed by qPCR, 24 h post-transfection (Figure S5A–C). The transfected cells either remained untreated or were treated with 20 µg/mL oxLDL for 24 has described earlier. The cells were rested for 4 days and restimulated with 10 µg/mL of Pam3cys. Additionally, the concentrations of IL-6 and TNFα in the supernatant were measured using ELISA.

Cell culture reagents and supplies were purchased from GIBCO BRL (Grand Island, NY); 25-hydroxycholesterol from New England Nuclear (Boston, MA). THP-1 and HepG2 cells were obtained from American Type Culture Collection (Rockville, MD). The reagents for real time RT-PCR were from AB Applied Biosystems (Warrington WA1 4 SR, UK). The chemicals used in this research were obtained from Sigma Chemical Co. (St. Louis, MO) or Bio-Rad Laboratories (Hercules, CA). Polyclonal rabbit antibodies against SREBP-1, SREBP-2, and HMG-CoA reductase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All solvents were obtained from Fisher (Fair Lawn, NJ) unless otherwise indicated. The enhanced chemiluminescence (ECL) reagents were from Amersham Biosciences (Piscataway, NJ). Oasis®Wax Cartridges were purchased from Waters Corporation (Milford, MA). The testosterone and 27-hydroxycholesterol were obtained from Research Plus Inc. (Bayonne, NJ). LK6 20×20 cm normal phase thin layer chromatography (TLC) plates were purchased from Whatman Inc. (Clifton, NJ).

Liver lysates were obtained in lysis buffer (RIPA buffer containing protease and phosphatase inhibitors). The lysate was centrifuged at 16,000× g at 4 °C for 15 min. The supernatant was transferred to a new microcentrifuge tube (1.5 mL) and sonicated for 15 s (cycle 0.5, amplitude 60%). Nuclear extracts from liver samples were prepared as described [10 (link)].
Protein concentration was determined using the bicinchoninic acid method, and 20–60 μg were loaded for western blot. The following antibodies were used: carbohydrate-responsive element-binding protein (ChREBP), glucose transporter 2 (GLUT2), sterol regulatory element-binding protein (SREBP) SREBP1 and SREBP2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Acetyl Coenzyme A carboxylase (ACC), phosphoenolpyruvate carboxykinase (PEPCK), total and phospho-(Ser473)-PKBAkt, AMP-activated protein kinase α2 (AMPKα2) and phospho-AMPKα2 (Thr172), ERK1/2 and phospho-p44/42 Erk1/2 (Thr202/Tyr204), mammalian target of rapamycin (mTOR) and phospho-mTOR (Ser2448) were provided by Cell Signaling (Beverly, MA, USA). Pyruvate carboxylase (PyC) antibody was raised at Salto’s laboratory. GAPDH (Sigma-Aldrich, Saint Louis, MO, USA) was used as a load control. Data were normalized using the values of the reference animals as 100%.