Fig. 3
Analysis of vRANK and fRANK expression in RAW264.7 cells and primary cultured macrophages by reverse transcriptase-PCR. (A) Upon treating RAW264.7 cells with PMA, Forskolin, and LPS to assess the inducibility of vRANK expression, PMA was found to induce vRANK expression. Time-course analysis of PMA treatment revealed that vRANK expression was observed at 9 h post-treatment, persisted for 24 h, and subsequently diminished while the expression level of GAPDH remained unchanged. (B) Induction of vRANK expression upon PMA stimulation was also observed in both spleen- and bone marrow-derived macrophages. (C) Treatment with TGF-β1 induced vRANK expression similarly to PMA, whereas BMP-2 treatment did not exhibit this effect. Investigation of the intracellular signaling pathways involved in PMA-induced vRANK expression using various inhibitors revealed that U0126, an MEK1/2 inhibitor, effectively blocked vRANK induction. (D) Knockdown of Sma68 was examined by treating cells with siRNA. The results showed that #1 (siRNA ID s20951) and #3 (siRNA ID s20953) each suppressed vRANK expression individually, as well as in combination while expression level of GAPDH unchanged.
