Western Blot Analysis of Protein Levels

The liver tissue samples and macrophage cells were lysed in radioimmunoprecipitation assay buffer (Millipore, Bedford, MA, USA) for total cell protein or in NE-PER extraction reagent (Thermo) for cytosolic and nuclear proteins. Concentrations of total protein were measured by Bradford protein assay reagents (Bio-Rad, Hercules, CA, USA). Equal amount of proteins was separated and then blotted in accordance with a previously described method [20 (link)]. Proteins on the membrane were blocked and then incubated with various primary antibodies followed by secondary antibodies (Table 2). Immunoreactive bands of target protein were detected using enhanced chemiluminescence solution (Bio-Rad). Each detected protein band was normalized by internal control proteins and was quantified using ImageJ software (version 1.53k).