Anti ldl receptor

Protein lysates were obtained by tissue homogenates in Polytron (MA099 Potter Unit, Marcone Equip., São Paulo, Brazil) by using buffer containing 20 mM Hepes, 150 mM NaCl, 10 % glycerol, 1 % triton, 1 mM EDTA, 1.5 mM MgCl₂ and protease inhibitors. An aliquot of supernatant was obtained after centrifugation and dissolved in SDS-glycerol. Equal amounts of sample protein were applied into a polyacrylamide gel and immunoblotting performed for SR-BI, LXR and the LDL receptor by using anti-SR-BI 1:1,000, anti-LXR 1 :1,000 (Novus Biologicals, Inc., Littleton, CO, USA), and anti-LDL receptor 1:1,000 (Santa Cruz Biotechnology Inc, USA). Membranes were incubated with HRP-conjugated antibody and reacted against ECL (Super Signal West Pico Chemiluminescent substract, Pierce, Rockford, IL, EUA). Nitrocellulose membrane stripping was done by washing with NaOH 0.8 mM. The difference between the bands was analyzed in pixels using the JX-330 Color Image Scanner (Sharp®) and ImageMaster software (Pharmacia Biotech). Results are expressed as arbitrary units corrected per β-actin (anti β-actin 1:1,000, Fitzgerald Industries International, Inc., Concord, MA). β-actin was utilized as a control and Ponceau staining of nitrocellulose membranes was also implemented to assure equal protein loading.

HepG2 cells were lysed in ice with Complete Tablet Buffer (Roche, Basel, Switzerland) supplemented with 2 mM sodium orthovanadate (Na3VO4), 1 mM phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA), 1:50 mix Phosphatase Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA), and 1:200 mix Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA). According to the standard protocol, 35 μg of protein of each sample was resolved on 10% SDS-PAGE gels, and polyvinylidene difluoride membranes (PVDF, GE, Healthcare Europe GmbH) was incubated overnight at 4 °C with the following specific primary antibodies: anti-LDL receptor (1:500; Santa Cruz, CA, USA), anti-pSRC (1:500; Santa Cruz, CA, USA), anti-SRC (1:500; Santa Cruz, CA, USA), anti-pAMPK (1:500; Santa Cruz, CA, USA), anti-AMPK (1:500; Santa Cruz, CA, USA) and anti-HMGCr (1:500; Santa Cruz, CA, USA). Protein expression was normalized and verified through anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). The results were expressed as means ± SD (% vs. control).