Anti phosphor p38

Letrozole (4,4-(1H-1,2,4-Triazol-1-ylmethylene) bisbenzonitrile) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Recombinant RANKL and M-CSF were obtained from PeproTech (Rocky Hill, NJ, USA), and the antibodies against NFATc1 and c-Fos were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The other antibodies (anti-phosphor-IKK, anti-IKK, anti-phosphor-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phosphor-p38, and anti-p38) were purchased from Cell Signaling Technology (Danvers, MA, USA). The leukocyte acid phosphatase kit (TRAP staining kit) and antibody for β-actin were obtained from Sigma-Aldrich. All other reagents were purchased from Sigma-Aldrich.

Western-blot analysis was performed as previous described [20 (link)]. Different primary polyclonal anti-iNOS, anti-Arg1, anti-IκBα (inhibitor of NF-κB), anti-phosphor-IκBα, anti-p65, anti-phosphor-p65, anti-p38, anti- phosphor-p38 and anti-tubulin antibody were all purchased from cell signaling technology (CST, USA) and used in the present study. After a secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse immunoglobulin (Ig) G antibody (1:10,000; SABC) incubated for 1 h at 37 °C, signals were detected by the enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA).