Western blot analysis

The procedure was followed by our past experiment [23, 24]. The expression of TLR-4 and associated signaling molecule proteins were detected by western-blot assay. The cells of liver tissue proteins (50 μg) from each sample were separated on SDS-PAGE, and then transferred to nitrocellulose filter membranes which were blocked overnight with 5% nonfat milk in TBST. Blots were probed over night at 4 °C with rabbit polyclonal TLR4 (diluted as 1:500), MyD88 (diluted as 1:500), TRIF (diluted as 1:500) and TRAF6 (diluted as 1:500) and then were followed by incubated with HRP-labeled secondary antibody for two hours before being washed 5–6 times in TBST. After further washing with TBST, ECL was added to identify the immunoreactive bands. The densitometry analysis of the immunoreactive bands was performed using the Fuji ultrasonic-doppler velocity profile (UVP) system and Image J program. The procedure was repeated for three times.