C fos

In this study, proteins were extracted using a protein extraction kit (Sangon Biotech), and the bicinchoninic acid (BCA) assay (Sangon Biotech) was used to determine the total protein content. After denaturation for 5 min, total proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA) via a constant current flow at 200 mA. Subsequently, the PVDF membranes were incubated with Bcl-2 (1:10,000), Bax (1:2000), Caspase-3 (1:5000), M-CSF (1:1000), RANKL (1:3000), OPG (1:3000), p-p65 (1:1000), p65 (1:1000), p-p38 (1:1000), p38 (1:5000), p-JNK (1:1000), JNK (1:1000), p-AKT (1:2000), AKT (1:2000), p-ERK (1:1000), ERK (1:1000), RANK (1:2000), NF-κB (1:1000), NFATc1 (1:10,000), and c-Fos (1:1000) antibodies (Abcam, Cambridge, MA) for 12 h at 4°C. TBS buffer was used to wash the PVDF membranes, and secondary antibodies (Abcam) were added and incubated at room temperature for 1 h. After the membranes were washed three times, chemiluminescent reagents were added, and the grayscale values of the bands were analyzed using ImageJ software. Each experiment was independently repeated 3 times. GAPDH was used to quantify the expression of various proteins.

Western blotting was performed using whole cell lysates. Aliquots of total protein (20-50 μg per lane) were electrophoresed on a 10% SDS-polyacrylamide gradient gel and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were incubated at 4 °C overnight with anti-GAPDH, MYC, c-Jun, p-c-Jun, c-Fos, p-c-Fos, JNK, p-JNK, or Igκ monoclonal antibody (all purchased from Abcam, Cambridge, MA). After rinsing in buffer wash, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX) diluted 1:10,000-30,000, followed by development with enhanced chemiluminescence reagents (Amersham, Little Chalfont, UK).

Slices were deparaffinized by Xylol, followed by rinse with phosphate-flushing fluid. After microwave-treated antigen retrieval, the samples were cooled below 35°C at room temperature. The slices were then incubated with 3% H₂O₂ at room temperature for 15 min. After that primary antibodies against P-P38 (1 : 100)/P-JNK (1 : 100)/C-fos (1 : 50)/γ-GCS-h (1 : 100) (Abcam, Cambridge, UK)/P-ERK (1 : 400)/C-jun (1 : 400) (Cell Signaling Technology, Boston, MA) were dripped into the samples and samples were incubated at 4°C for 24 h. After reheating at 37°C for 1 h, the respective secondary antibodies were added for incubation at 37°C. DAB coloring was then performed, and cell nucleus was redyed with Harris hematoxylin. The samples were dehydrated until becoming transparent and were sealed with neutral balsam. The slices were observed under a light microscope. Five different random views of each slice were observed. A semiquantitative analysis on integrated optical density (IOD) was conducted by HIS-IPP (Image Pro Plus 6.0) software.