Western Blot Analysis of PPAR-γ and NF-κB Signaling

Tissues of inflamed joints were separated and homogenized on ice in cold lysis buffer (Beyotime, China) plus 1:100 volume of phenylmethyl sulfonylfluoride (PMSF). RAW 264.7 cells were treated with LPS (1 μg/ml) and Rg1 (12.5 μM, 25 μM, and 50 μM) for 24 h and then washed with cold PBS and lysed in ice-cold lysis buffer (Beyotime, China) plus 1:100 volume of PMSF. The supernatant was aliquoted and protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Proteins were segregated by electro-blotted and SDS-PAGE into a membrane of nitrocellulose. The blots were probed with antibodies against PPAR-γ, IκBα, p-IκBα, NF-κB p65, p-p65 (all antibodies were diluted by 1:1000) at 4°C overnight and later incubated with horseradish peroxidase-conjugated IgG secondary antibody (Beyotime, China; 1:5000 dilution) for 1 h. The expression of every protein was detected by the detection system of ECL (ChemiDoc^™XRS, Bio-Rad, Shanghai, China). The respective bands were quantitated and images were collected utilizing Quantity One Software (Bio-Rad). The fold increase over control was used to express the results [42 (link)].