Gapdh

Whole cell extract and nuclear and cytoplasmic proteins were prepared from RAW264.7 cells by using a protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The protein concentration was measured by the Bicinchoninic acid (BCA) method. Samples (20 µg total proteins) were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon P; Millipore, MA, USA). After blocking with 5% nonfat milk in phosphate buffer solution with tween-20 (PBST) for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibody and then incubated with the conjugated secondary antibodies. The membranes were incubated with Electro-Chemi-Luminescence (ECL) detection kits for 1 min and then exposed to X-ray film. The intensity of the immunoreactive bands was determined using a densitometric analysis program (Image Gauge V3.12; Fuji Photo Film, Tokyo, Japan). Mouse monoclonal to IRF5, GAPDH and PCNA mAbs were purchased from Abcom company (Cambridge, MA, USA). Goat anti-mouse IgG was purchased from Zymed Laboratories (San Francisco, CA, USA).

CORM-3 and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Collagen Type I Alpha 1 (COL1A1), Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN) antibodies were purchased from Cell Signalling Technology (Denver, MA, USA). Cytochrome C (Cyto-C), Bax, cleaved-caspase3, and Bcl-2 were purchased from Proteintech (Chicago, IL), whereas nuclear factor erythroid 2-related factor 2 (Nrf2), HO-1, NAD(P) H:quinone oxidoreductase 1 (NQO1), β-actin, Lamin B, and GAPDH antibodies were purchased from ABCom (Cambridge, MA). MEM Alpha Modification (α-MEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Life Technologies (Rockville, MD). All reagents used in this study were of analytical grade or met the criteria required for cell culture experiments.