Figure 3.

c-Abl^−/− osteoblasts show enhanced Erk1/2 activation in response to BMP2, which contributes to p16^INK4a upregulation. (a) Mutant and WT cells were cultured as in Fig. 1c and one plate was collected at day three of each passage. Passage ‘o/n’ stands for cells that have been grown overnight. Erk activation was analysed by western blot. As p-Erk1/2 levels are similar in different passages of WT or c-Abl^−/− osteoblasts, an average is shown in the right panel. (b) Inhibition of Mek1/2 activation decreased the levels of p16^INK4a. Osteoblasts (passage 2) were treated for 24 h with different concentrations of U0126 and western blot analysis was used to determine the protein levels of p16^INK4a. Right panel: quantification of p16^INK4a. (c) c-Abl deficiency altered BMP2-induced Erk1/2 activation. c-Abl^−/− and control osteoblasts were stimulated with 100 ng ml⁻¹ BMP2 for different periods of time and western blot analysis was used to determined the activation of these kinases. Right panel: quantification of active Erk1/2. (d) Blockade of BMP2 action with noggin/chordin led to Erk1/2 activation. c-Abl^−/− and WT osteoblasts were treated with BMP2 (B; 100 ng ml⁻¹) or noggin/chordin (N/C; 0.5 μg ml⁻¹ and 1.0 μg ml⁻¹, respectively) for 2 days and western blot was used to determine the activation of Erk1/2 and Tak1. Note that p-Tak1 is difficult to detect in WT cells. Right panel: quantification. Data are means ± s.e.m. (n = 3) *P < 0.05, compared to untreated cells or WT cells. Uncropped images of blots are shown in Supplementary Fig. S8.