Enhanced chemiluminescence plus detection system

Equal amounts (80 μg) of protein samples were resolved by 10% SDS-PAGE and then electrotransferred to nitrocellulose membranes (Millipore). All membranes were incubated overnight at 4°C with anti-P-HMGCR (1 : 1000, Millipore), anti-HMGCR (1 : 1000, Upstate), anti-P-AMPK (1 : 2000, CST), anti-AMPK (1 : 1000, CST), and anti-β-actin (1 : 10000, Abcam). After incubation with secondary antibody (Zsbio, Ltd., China), immune complexes were detected using the enhanced chemiluminescence plus detection system (Amersham). Immunoreactive bands were quantified using the Alphaimager 2200. The relative target protein levels were normalized to β-actin.

Equal amounts of protein from different samples were subjected to 10% SDS-PAGE followed by electrotransfer from the gel to polyvinylidene difluoride membranes (Millipore). The membranes were incubated overnight at 4 °C with anti-CYP7A1 (1:200, Santa Cruz) and anti-HNF-4α (1:1000, Invitrogen) antibodies. β-actin protein was evaluated as a loading control (1:10000, Abcam) for cytoplasmic protein and Lamin B1 protein as a loading control (1:2000, CST) for nuclear protein. Immune complexes were detected using the Enhanced Chemiluminescence Plus Detection System (Amersham).