Tbs t

For protein analysis 50 mg of liver or intestine were homogenized in 700 μl of RIPA buffer containing 1% protein inhibitor cocktail (Santa Cruz, USA) and 1 μM PMSF, then centrifuged (14000 rpm at 4°C for 15 minutes). Protein concentration of the samples was measured in a Nano Drop spectrophotometer and 120 μg of protein was loaded on a 12.5% SDS-PAGE gel. After electrophoresis, proteins were transferred to a PVDF (Roche Applied Science) membrane. The Membrane was blocked with 3% skim milk in Tris-buffered saline containing Tween-20 (TBS-T) (Roche Applied Science) for 2 hrs at room temperature. The membrane was washed (three times, 15 min each) in TBS-T and then incubated with primary antibody for 1.5 hrs with anti rabbit polyclonal antibody LXR (1:300 dilution, Novus Biological (NB400-157)). After 3 washes in TBS-T, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:10,000 dilutions, Roche Applied Science) at room temperature for 1.5 hrs. The membranes were washed and exposed to ECL western blotting detection reagents (Roche Applied Science) and then exposed to films for 30s to 1 minute. Films were developed, scanned, and band densities were measured with Lab Work analyzing software (UVP, UK). Data are expressed as the percent ratio of the protein bands to β- Actin band [10 ,13 (link),16 (link)].

Cells were harvested in LIPA buffer (Sigma) as previously reported^{13 (link)}. After centrifugation at 15,000 × g for 5 min, the supernatant was boiled for 5 min in SDS-PAGE sample buffer (Wako) and loaded onto 5–20% gradient or 15% gels for 1~2 h. Proteins were transferred to nitrocellulose membranes (Bio-Rad) by using the Trans-Blot Electrophoretic Transfer cell (Bio-Rad). The membrane was washed in 20 mM Tris-HCl buffer with 1% Tween 20 (TBS-T) (Sigma-Aldrich), incubated for 1 h in blocking buffer (5% milk powder (Merck) in TBS-T), and then further incubated overnight with primary antibodies; Anti-LC3 (Cell signaling) and anti-GAPDH (Santa Cruz) antibodies were purchased and diluted in blocking buffer by 1:3000. The horseradish peroxidase (HRP)-labeled secondary antibody (Dako) was diluted by 1:5000 in TBS-T for secondary antibody staining for 1 h at RT. The membrane was then washed in TBS-T and the signals were developed using the Lumi-Light reagent (Roche) for 5 min. The signal was detected on the LAS-3000 imaging system (Fuji).