FIGURE 5.

Both Smad1/5 and Erk activation are required for Runx2 stabilization. A–C, Erk activation further enhanced Smad1/5-induced Runx2 stabilization, whereas U0126 blocked it. 293T cells were transiently transfected with Myc-Runx2, FLAG-Smad1, FLAG-Smad5, HA-p300, and MEK1 expression vectors as indicated and incubated for 24 h. When indicated, cells were treated with U0126 for the last 2 h. IP and/or IB analysis was then performed. AcK, anti-acetylated lysine antibody. D, knockdown of Smad1/5 decreased both the basal and Erk-induced Runx2 protein levels. 293T cells were transfected with siRNAs for Smad1 and -5 (siSmad1/5) and a non-targeting control siRNA (siControl), and a second transfection with Myc-Runx2 and MEK1 expression vectors was performed 6 h later. After an additional incubation for 32 h, IP and IB analysis was performed.