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. 2012 Mar;165(5):1584–1594. doi: 10.1111/j.1476-5381.2011.01664.x

Figure 6.

Figure 6

Enhancement of RUNX2 activity by TM-25659 depends on TAZ expression. (A) Confluent MC3T3-E1 cells were treated with TM-25659 for 48 h under osteoblast differentiation conditions. Nuclear proteins were harvested and analysed by SDS-PAGE and immunoblot of RUNX2. (B and C) 293T cells were transfected with both RUNX2 and Flag-tagged TAZ expression vectors, and treated with either vehicle or TM-25659 (50 µM) for 24 h. Cellular proteins were incubated with either Flag-M2 agarose beads (B) or biotinylated RBE DNA, followed by incubation with streptavidin agarose beads (C). Precipitates were resolved by SDS-PAGE and protein blots were incubated with antibodies against RUNX2 or TAZ. (D) 293T cells were transfected with RUNX2 and/or TAZ expression vector along with reporter genes (6xOSE-luc and pCMVβ), followed by treatment with TM-25659 (50 µM) for 24 h. (E) TAZ WT and KO MEF cells were transiently transfected with RUNX2 expression vector and reporter genes (6xOSE-luc and pCMVβ). Cells were treated with TM-25659 (50 µM) for 24 h and relative reporter activity was expressed as a fold induction after normalization with β-galactosidase activity. *P < 0.05; **P < 0.005.