Anti ikk

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.

For the subcellular fractions, primary microglia-enriched cultures were lysed in hypotonic lysis buffer and incubated on ice for 30 min, and then subjected to homogenization. The lysates were loaded onto a sucrose gradient in lysis buffer, and the supernatant above the sucrose gradient was performed as the cytosolic fraction. The pellet was solubilized in hypotonic lysis buffer and was applied as the membranous fraction. For the whole cell lysate extraction, cultures were washed with cold phosphate-buffered saline (PBS) and lysed with cell lysis buffer. The lysates were incubated on ice for 30 min and then centrifuged at 12,000×g for 25 min. Protein levels were quantified via bicinchoninic acid (BCA) assay. Membranes were blocked with 5% non-fat milk and then incubated with the following primary antibodies: β-actin, TH, ionized calcium-binding adapter molecule-1 (Iba-1), α-synuclein, p47, p67, gp91, BDNF, GDNF (Abcam, Cambridge, UK), phospho-p65 (p-p65), p65, phospho-IKK (p-IKK), anti-IKK, phospho-p38 (p-p38), p38, phospho-JNK (p-JNK), JNK (Cell Signaling Technology, MA, USA), and horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories, CA, USA). All the antibodies were diluted between 1:200 and 1:1000. The blots were developed with the enhanced ECL reagent.

Total proteins were extracted with lysis buffer (10 mM Tris-HCL, 1 mM EDTA, 1% sodium dodecyl sulfate, 1% Nonidet P-40, 1:100 proteinase inhibitor cocktail, 50 mM b-glycerophosphate, and 50 mM sodium fluoride). Aliquots of 20–60 mg per sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to the polyvinylidene fluoride membranes and blocked with 5% nonfat milk powder in PBST (PBS with 0.1% Tween). Next, they were incubated with the following primary antibodies overnight: anti-Osterix, anti-Runx2, anti-ALP, anti-P65, anti-p-P65, anti-IKBα, anti-IKK, anti-p-IKK (Cell Signaling Technology). The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Boster, Wuhan, China). The blots were visualized using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s recommended instructions.