*4.5. Immunoblotting*

To determine the expression of intracellular proteins, immunoblotting was performed with the aforementioned antibodies. For NFATc1 and Nrf2 expression, RAW264.7 cells were seeded onto a 6 well plate (2.0 × 10<sup>4</sup> per well) in α-MEM containing 10% FBS. On the following day, 0, 1, 2.5, and 5 μM FX were added to the culture medium, and cells were grown under sRANKL or TNFα/IL-6 stimulation for 4 days. For MAPK, p65, and PI3K expression, RAW264.7 cells were cultured in α-MEM containing 10% FBS with 0, 1, 2.5, and 5 μM FX for 4 days. Thereafter, cells were incubated with sRANKL or TNFα/IL-6 in serum-free media for 30 min.

Total cell lysates were obtained using cold radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1% NP-40; 1% sodium deoxycholate; 0.1% SDS). The crude extract was separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Protein bands were detected using an enhanced chemiluminescence system (Amersham Biosciences, Little Chalfont, UK). Nuclear extracts were prepared with the NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. The relative expression of each protein was determined by densitometric analysis using ImageJ software.

#### *4.6. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Real-Time PCR*

The expression levels of osteoclast-related genes were measured using RT-PCR with specific primers. cDNA and target-specific primers were added to the power SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA). PCR cycling parameters were as follows: amplification (1 cycle at 50 ◦C for 2 min, 1 cycle at 95 ◦C for 10 min, and 40 cycles at 95 ◦C for 15 s and 60 ◦C for 1 min). Fold changes of gene expression were calculated with the ΔΔCt method using ribosomal protein S18 as the reference gene. Specific murine primers are summarized in Table 1.


**Table 1.** Oligonucleotide primers used for RT-PCR.
