Homogenization and preparation of tissue extracts and western blotting

Liver tissue (30-50 mg) was homogenized in ice-cold lysis buffer containing 0.5 % CHAPS, 10 mM Tris-HCl pH 7.5, 1 mM MgCl₂, 1 mM EDTA, 10 % glycerol and protease and phosphatase inhibitors (5726, P0044, P8340, Merck-Sigma Aldrich) using the Brinkman PT 10/35 Polytron. Extracts were then incubated for 45 min at 4° C with continuous agitation. Liver extracts were cleared by centrifugation at 13000 x g for 15 min at 4° C. Protein determination was performed by the Bradford dye method, using the Bio-Rad reagent and bovine serum albumin (BSA) (Merck-Sigma Aldrich) as the standard. Absorbance was measured in a Synergy HT plate reader at 595 nm.

Cytosolic and nuclear extracts were isolated from 25 mg of tissue as follows. Livers were disrupted in 0.3 M sucrose solution containing the protease and phosphatase inhibitors mentioned above by using a micro pestle. Homogenates were centrifuged at 1000 x g for 10 min at 4° C to separate the cytosolic fraction (supernatant) and the nuclear fraction (pellet). Supernatants containing the cytosolic fraction were centrifuged 3 times at 27000 x g for 15 min at 4° C, and transferred to a new tube for protein analysis. Pellets containing the nuclear fraction were washed with 0.3 M sucrose solution and centrifuged at 1000 x g for 10 min at 4° C. After three washing steps, pellets were then resuspended in RIPA buffer (Merck-Sigma Aldrich) and incubated for 1 h at 4° C on a rotating mixer. After sonication, cellular debris was removed by centrifugation at 8000 x g for 15 min at 4° C and the supernatants containing the nuclear fraction were collected. Protein determination was performed using the Pierce BCA Protein Assay Kit (Thermofisher Scientific) and a standard curve of BSA. Absorbance was measured in a Synergy HT plate reader at 562 nm.

For protein analysis, protein extracts (30 μg) were boiled for 5 min with Laemmli buffer (200 mM Tris-HCl pH 6.8, 5.2 % SDS, 40 % glycerol, 3 % β-mercaptoethanol, and 0.08 % bromophenol blue) and then separated by 8-12 % SDS-PAGE (80 V-120 V, 2 h). Proteins were transferred to a polyvinylidene fluoride membrane (PVDF) using a semi-dry method (Trans-blot SD SEMI-Dry Electrophoresis Transfer Cell, Bio-Rad; 25 V and 1.5 A, 37 min). Membranes were blocked using 5 % non-fat dried milk in PBS for 1 h at RT and then incubated with primary antibodies in PBS (Table 2) overnight at 4° C. After 3 washes with PBS containing 0.1 % Tween-20, membranes were incubated with the corresponding peroxidase-conjugated secondary antibodies (anti-rabbit, 1/5000, Bio-Rad; anti-goat, 1/50000, Merck-Sigma Aldrich) for 1 h at RT. Immunoreactive bands were visualized using the ECL Western blotting protocol. Blots were normalized by lane charge using antibodies against GAPDH, VINCULIN or LAMIN B, as indicated in each figure. Different exposure times were used for each membrane to ensure the linearity of the intensity of the bands. The densitometric analysis was carried out with the ImageJ program and was expressed in arbitrary units.