Mammalian nuclear and cytoplasmic protein extraction kit

Total protein was extracted with NE-PER nuclear and cytoplasmic extraction reagents (Thermo). Nuclear proteins were isolated with a mammalian nuclear and cytoplasmic protein extraction kit (TransGen Biotech). Next, 50–100 μg aliquots of protein were separated on 10% polyacrylamide-SDS gels (Pplygen) and transferred to Immobilon™-P membranes (Millipore). After blocking with TBS/5% nonfat dry milk (Pplygen) for 1 h, the membrane was incubated with antibodies against mouse PPARγ (Cell Signaling Technology), histone H3 (Bioworld), and β-actin (Sigma) overnight at 4 °C. Next, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Pierce, Malibu, CA, USA) for 1 h at room temperature. Antibody binding was visualized with an enhanced chemiluminescence kit, according to the manufacturer’s protocols (Pierce).

Lysates were prepared from cultured RA-FLS as previously described [17 (link)]. Cell nuclear and cytoplasmic extracts were prepared using the Mammalian Nuclear and Cytoplasmic Protein Extraction Kit (TransGen Biotech, Beijing, China) following the manufacturer’s protocol. Protein concentrations were measured using a Quick Start™ Bradford Protein Assay (Bio-Rad, USA).
Western blot assays were performed as previously described [16 ]. Immunoreactive bands were visualized using the Enhanced Chemiluminescence Detection Kit (Invitrogen, USA). Image J software was used to measure the intensity of each band. The relative level of each protein of interest was normalized to the endogenous β-actin, GAPDH or lamin B1 in each experiment.