Tissue protein extraction reagent

All the chemicals used in the current study were of analytical grade. Primary antibodies (Nrf-2, HO-1, PSD95, Phosphorylated c-Jun N- Kinase, SYP, β-actin) Sodium dodecyl sulfate (SDS), Scopolamine (Santa Cruz, CA, USA), Tetra methylene diamine (Temed), Ammonium per sulfate (APS) (Daejung Chemicals and Metals Co. Ltd, Korea), Tissue protein extraction reagent (T-PER), Methanol (Sigma Aldrich, Burlington, MA, United States), Skim Milk Powder (Oxoid Ltd, Basingstoke, UK), RNA wait (Beijing Solar bio Science and Technology Co. Ltd), Enhanced Chemiluminescence (ECL) solution A (Luminol) and solution B (Peroxide solution) and secondary antibody (anti-mouse) (Bio-Rad, United States) were bought while folicitin was gifted by Dr. Umar Farooq.

BMP-2/CPC particles at different doses were subcutaneously transplanted to C57BL/6 mice. Three days afterward, the particles and the surrounding tissues were collected. The 0 mg/ml BMP-2/CPC group served as a control. The tissue samples were incubated in 1 ml of tissue protein extraction reagent (Sigma-Aldrich, USA) containing protease inhibitors (1 tablet of protease inhibitor cocktail [Roche] for 10 ml) and homogenized with a tissue homogenizer. Tissue lysates were incubated for 1 h at 4 °C and centrifuged at 5000 g for 5 min. Cytokines were detected by ELISA (Mouse IL-1β and Mouse IL-1Ra, R&D Systems, USA) [31 ].