Anti hnf 4α

HNF-4α phosphorylation was assayed according to the method previously described22 (link). The tissue powder and cells were lysed in RIPA buffer containing 1% PMSF and 1% phosphatase inhibitor cocktail. The protein concentration was adjusted to 1 μg/μl. The anti-HNF-4α antibody (mouse monoclonal antibody, Invitrogen) was covalently crosslinked onto Protein A/G resin using a Crosslink Immunoprecipitation Kit (Pierce, USA).A total of 500 μg of protein was incubated overnight at 4 °C with the crosslinked antibody. The HNF-4α protein was eluted from the resin without antibody contamination and then subjected to SDS-PAGE on a 10% gel and transferred to a nitrocellulose membrane (Millipore). Phosphor-HNF-4α and HNF-4α proteins were detected using anti-phosphoprotein (Invitrogen) and anti-HNF-4α (Invitrogen) antibodies, respectively.

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).

Liver tissues were lysed in RIPA lysis buffer with protease inhibitors and phosphatase inhibitors (Bimake, Houston, USA) for total protein according to the manufacturer’s instructions (Shenergy Biocolor Bioscience & technology CO., Shanghai, China). Protein concentration was measured using BCA Protein Quantitative Assay Kit (Shenergy Biocolor Bioscience & technology CO., Shanghai, China). The target proteins were blotted with the following antibodies: anti-HNF4α (41770, Invitrogen, USA), anti-SIRT1 (sc-15404, Santa Cruz Biotechnolosy, USA), anti-PPARα (MAB3890, Merck KGaA, Germany), anti-GAPDH (60004–1, Proteintech, China). The relative target protein levels were quantified by densitometry normalized to GAPDH on the same membrane. The samples in one group from three different mice and three independent experiments were performed.