Luciferase activity was measured using a Promega Luciferase Assay System (Promega). The activity was measured using a Fluoroskan® Ascent FL (Thermo Fisher PF-6463922 Scientific, Rochester, NY, USA). The cells were cotransfected with pRL-TK as an internal control to normalize the reporter gene activity and ensure the expression of luciferase in all subsequent experiments. Western blot analysis RAW 264.7 cells were incubated with or without RANKL in the presence or absence of kinsenoside. The extraction of cytoplasmic and nuclear proteins was performed as described
previously [24]. The primary antibodies were obtained from the following sources: p65, phosphorylated p65 (p-p65), IκBα, phosphorylated IκBα (p-IκBα), IKKα, BIBW2992 IKKβ, and phosphorylated ΙΚΚα/β (p−ΙΚΚα/β) from Cell Signaling (Danvers, MA, USA), and proliferating cell nuclear antigen (PCNA), α-tubulin, p50, and NFATc1 from Santa Cruz (CA, USA). The whole-protein extracts prepared following the method described by Lee et al. were used for the Western
blot analysis of NFATc1 expression [25]. Western blot analysis was performed as described previously [17]. IKK activity assay IKK activity was measured by an IKKα KinEASE™ FP Fluorescein Green Assay Kit (Millipore, Billerica, MA, USA) following the manufacturer’s instructions. A fluorescence polarization assay was performed using a Synergy 2 fluorescence plate reader (BioTek Instruments, Inc., USA) with excitation set at 485 nm and emission at 530 nm. RT-PCR analysis The BMs were cultured for 3 days in the presence of M-CSF (20 ng/ml). Adherent cells were used as osteoclast precursors. To generate osteoclasts, osteoclast precursors were cultured with M-CSF (20 ng/ml) and RANKL (50 ng/ml) for 3 days in the presence of kinsenoside. Total RNA was extracted
with TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The PCR primer sequences for mouse ALP, cathepsin K (CAK), dendritic cell-specific transmembrane protein (DC-STAMP), MMP-9, RANK, TRAF6, and TRAP were as follows: primers for ALP were 5′-GTATGCCTCCTGCATTGGGG-3′ (sense) and 5′-TGTTCCTGCTGGAAGTTGCC-3′ (antisense); primers for CAK were 5′-CTGCCCATAACCTGGAGG-3′ (sense) Aprepitant and 5′-GCCCTGGTTCTTGACTGG-3′ (antisense); primers for DC-STAMP were 5′-ACCCGTTGCCCTGCTCTCTT-3′ (sense) and 5′-ACGGAGGCCACACGACAGAA-3′ (antisense); primers for GAPDH were 5′-CTTCATTGACCTCAACTACATGGTCTA-3′ (sense) and 5′-GATGACAAGCTTCCCATTCTCAG-3′ (antisense); primers for MMP-9 were 5′-GGTCTAGGCCCAGAGGTA-3′ (sense) and 5′-GGTCGTAGGTCACGTAGC-3′ (antisense); primers for RANK were 5′-GTGACTCTCCAGGTCACTCC-3′ (sense) and 5′-GGCAGACACACACTGTCG-3′ (antisense); primers for TRAF6 were 5′-GTTCTCAGGGAGCCCTAC-3′ (sense) and 5′-GAGGCACAGCTAAGGGAC-3′ (antisense); primers for TRAP were 5′-GAACCGTGCAGACGATGG-3′ (sense) and 5′-GGAAGTTCCAGCGCTTGG-3′ (antisense).