Real Time PCR for ABCB11 and few NRs

Real Time PCR: Total RNA was isolated using NucleoZOL (Takara Cat. No. 740404.200) following manufacturer's instruction. cDNA was prepared from (deoxyribonuclease treated) total RNA using RevertAid Reverse Transcriptase (Thermo Cat. No. EP0441) following the 'manufacturer's instructions. Real Time PCR was done with unique oligonucleotide primers targeting ABCB11 gene using GoTaq® qPCR Master Mix (Promega Cat. No. A6001) following 'manufacturer's instructions on a Veriti Thermo Cycler from Applied Biosystems Waltham, Massachusetts, USA and data was acquired using the software platform associated with the same machine. Western Blot: Total proteins from HepG2 cells were prepared and run on 10% SDS-PAGE and transferred to a PVDF membrane using a transfer apparatus following the standard protocols (Bio-Rad). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 1h the membrane was washed once with TBST and incubated with antibodies against human ABCB11 (Affinity, Catalog #DF 9278) 1: 2000 dilution; human β-actin (Santa Cruz Cat.# SC4778), dilution 1:1000. The membrane was washed and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-rabbit (Santa Cruz Cat# SC-2004)/anti-mouse antibodies (Cat.#SC-2005) for 2 h. Blots were washed with TBST four times and developed with the ECL system (Bio-Rad, US Cat.#170-5060) according to the 'manufacturer's protocol.