Protein Expression Analysis in Rat Tissues

Briefly, rat liver, kidney, brain, and heart tissue samples were lysed in RIPA buffer supplemented with freshly added protease and phosphatase inhibitor cocktail 1:100 (Sigma), and protein concentration was determined by Bradford assay⁷⁰ . The previously developed procedures with slight modifications were used to perform SDS-PAGE and western blot investigations^{71 (link),72 (link)}. For SDS-Page, equal amounts of proteins were loaded using BIO-RAD Mini protein TGX precast gels on the bio-rad Mini protein tetra system (10,025,025 REVA). After protein separation by gel electrophoresis, the proteins were transferred to PVD membranes using bio-rad trans blot Turbo transfer system. PVD membranes (bio-rad) were blocked for 2 h with either 5% BSA (Sigma) or 5% nonfat dry milk (Bio-Rad, Cat. #170–6404) then incubated with primary antibody overnight at 4 °C. The following antibodies were used: NFκB p65 (ThermoFisher Scientific Catalog # 2A12A7), STAT3 (Thermofisher Scientific Cat. # MA1-13,042), COX-2 (Invitrogen Cat. # PA1-9032), IL-1 (Abcam Cat. # ab2105), TNF-α (Abcam Cat. # EPR19147) and β-actin (Abcam Cat. # ab49900). The antibodies signals were detected by ECL western blotting substrate (bio-rad) and blots were visualized using the bio-rad Gel documentation system (ChemiDoc MP System).