Quantitative Western Blot Analysis of Liver Proteins

Liver samples were homogenized in lysis buffer using a Potter-Elvehjem homogenizer, and total protein and nuclear extracts were prepared as described previously [16 (link)]. Protein content was determined by the Bradford assay [31 (link)]. Western blots were performed using four samples per group, each sample pooled from two animals. A total of 20–30 µg of protein extracts was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were then transferred onto Immobilon polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA) and blocked for 1 h at room temperature with a 5% non-fat milk solution in Tris-buffered saline (TBS) containing 0.1% Tween-20. Membranes were then incubated with specific primary antibodies (see the list of antibodies used in the Supplementary Material, Table S2). Detection was performed using the Immobilion Western HRP substrate Peroxide Solution^® (Millipore, Billerica, MA, USA). To confirm the uniformity of protein loading, blots were incubated with antivinculin antibody (Santa Cruz Biotech, Dallas, TX, USA) as a control for total protein extracts, and with anti-TBP antibody (AbCam, Cambridge, UK) as a control for nuclear protein extracts.