Polyclonal goat anti rabbit hrp secondary antibody

Monoclonal rabbit phospho-Smad2 antibody (Cell Signaling, Boston, MA), monoclonal rabbit phospho-Smad3 antibody (Cell Signaling) and monoclonal rabbit phospho-NF-kB (Cell Signaling) were used (overnight at 4 °C) for LX-2 and KUP5 cells. Thereafter, polyvinylidene fluoride (PVDF) membranes were incubated with polyclonal goat anti-rabbit HRP secondary antibody (Agilent Technologies, Santa Clara, CA) at room temperature for 1 hour. Monoclonal rabbit total Smad2 and Smad3 antibodies (Cell Signaling) for phosphorylated Smads, and β actin (Cell Signaling) for phosphorylated NF-kB were used as loading controls.

Liver tissue samples were homogenized in approximately 0.5 ml of ice-cold lysis buffer and 1 × PhosSTOP Phosphatase Inhibitor Cocktail Tablets solution (Hoffmann-La Roche) and then centrifuged at 72,000 × g using Optima TLX ultracentrifuge (Beckman Coulter) for 1 hour at 4 °C. After centrifugation, the resultant supernatant was kept as cytosolic p67^phox subunit samples, whereas the pellet was resuspended and sonicated in 0.5 ml ice-cold lysis buffer and kept as membrane p67^phox subunit samples. Liver protein samples (~50 μg) from all mice were subjected to western blotting. Polyclonal rabbit p67^phox antibody (Sapphire Bioscience, Australia), monoclonal rabbit phosphorylated and total p38 antibody (Cell Signaling) and polyclonal rabbit phosphorylated and total extracellular signal-regulated kinase (Erk1/2) antibody (Cell Signaling) were used to probe liver protein extracted from BDL and Mdr2-KO mouse livers. Thereafter, PVDF membranes were incubated with polyclonal goat anti-rabbit HRP secondary antibody (Agilent Technologies). Total p38, total Erk1/2 were used as loading controls.
Blots probed with protein from LX-2 cells and the livers of BDL and Mdr2-KO mice were developed in enhanced chemiluminescence reagent (Thermo Fisher Scientific). Intensities of the digitally detected bands were evaluated densitometrically using Gel Doc XR System (BioRad, Hercules, CA).