Goat anti mouse or goat anti rabbit igg

Protein concentrations in the cell, nuclei, and cytoplasmic extracts were measured using the Protein Assay Kit 2 according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA, USA). Thirty micrograms of proteins from the cell or nuclei fractions were separated by SDS-polyacrylamide gel electrophoresis using 10% polyacrylamide gel. Proteins were then transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore, MA, USA). The membranes were blocked overnight using 5% nonfat milk in 50 mM Tris/150 mM HCl (pH 7.5) containing 0.1% Tween 20 (TBS/Tween). After three 5-min rinses with TBS/Tween, membranes were probed with polyclonal anti-PPARγ, anti-COX-2, anti-p65 NF-κB, or anti-IκBα (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h at room temperature. After three 5-min rinses with TBS/Tween, horseradish peroxidase- (HRP-) conjugated secondary antibodies goat anti-rabbit or goat anti-mouse IgG (Santa Cruz Biotechnology) were applied for 1 h at room temperature. Protein bands were visualized using a chemiluminescence detection system (Immun-Star HRP; Bio-Rad Laboratories, Hercules, CA, USA). To normalize protein signals, stripped PVDF membranes were reprobed with monoclonal anti-β-actin (Sigma-Aldrich) for cytosolic and cell fractions or with polyclonal anti-lamin A/C (Santa Cruz Biotechnology).