Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPAR? and Fatty Acid Uptake

Labrie, Marilyne; Lalonde, Simon; Najyb, Ouafa; Thiery, Maxime; Daneault, Caroline; Des Rosiers, Chrisitne; Rassart, Eric; Mounier, Catherine

Tissues or cultured cells were homogenized in cold lysis buffer (50 mM TrisÁHCl pH 7.3, 150 mM NaCl, 5 mM EDTA, 0.2% Triton X-100, 2 mM sodium orthovanadate and 10% Complete protease inhibitor). Lysates were then incubated 30 min at 4°C, cleared by centrifugation and stored at -80°C until further use. Based on Bradford assay [46] , 50 μg of protein of each sample were separated on SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% milk, 1h at room temperature, the membranes were incubated with the primary antibodies overnight at 4°C. Dilutions of the primary antibodies were: 1:1000 for PPARγ (C26H12), 1:1000 for total AMPKα antibody; 1:1000 for phospho-AMPKα (Thr172) (40H9) antibody; 1:1000 for ACC antibody; 1:300 for anti-phospho-ACC (Ser79) antibody; 1:5000 for Plin2 antibody, 1:10000 for H-apoD antibody [43] , 1: 1000 for the mouse apoD (m-apoD) antibody, 1:10000 for HPRT antibody and 1:100000 for β-actin antibody. Primary antibodies were then detected with a goat anti-rabbit horseradish perioxidase-conjugated secondary antibody (1:10000) and visualized by chemiluminescence. Amidoblack staining was used as loading control. Briefly, membranes were stained for 20 min in amidoblack solution (0,1% Amidoblack, 40% v/v methanol and 10% v/v acetic acid) and washed 10 min twice in decoloration solution (40% v/v methanol and 10% v/v acetic acid). Bands were quantified by densitometry using the image J software.