AS extract suppresses the mRNA expression of c-Fos, c-Jun, NFATc1, OSCAR and TRAP in BMMs treated with RANKL. (A) BMMs were pretreated with or without AS extract (50 mg/l) for 1 h and with RANKL (100 ng/ml) for the indicated time. Total RNA was isolated using TRIzol, and 1 μg of total RNA was used to transcribe cDNA. cDNA (1 μl) was used as a template for real time RT-PCR. The mRNA expression of the indicated genes was analyzed by real time RT-PCR. (B) AS extract inhibits the expression of c-Jun, c-Fos and NFATc1 induced by RANKL. BMMs were pretreated with or without AS extract (50 mg/l) for 1 h and were treated with RANKL (100 ng/ml) for the indicated time. Cells were lysed in the lysis buffer, and lysates were analyzed by Western blotting with antibodies against c-Jun, c-Fos, NFATc1 and actin. The intensities of the protein bands were analyzed and normalized to actin. Similar results were obtained in at least 3 independent experiments.