Figure 2. ERK and p38 MAP kinase stimulation of RUNX2 phosphorylation and transcriptional activity.
A. MEK1 and MKK6 dose-response. COS7 cells were transfected with wild type RUNX2 expression vector, 6OSE2-luc reporter and increasing amounts of either constitutively active MEK1 (Meksp) or MKK6 (Mkk6sp) expression vectors (0.05, 0.1, 0.25, 0.5, 0.75, 1.0 μg/dish) (upper panel). Firefly luciferase activities were normalized to renilla pyriformis luciferase plasmid. Results are presented as normalized luciferase activity (F/r) and fold-stimulation relative to a LacZ control transfection. RUNX2-S319-P, P-ERK1/2 and P-p38 were detected by Western blotting using the indicated antibodies (lower panel). B. Immunofluorescence detection of endogenous RUNX2 phosphorylation in MC3T3E1 preosteoblast cells. MC3T3-E1 clone 4 cells were transfected with control (LacZ), Meksp or Mkk6sp expression vectors and stained with P-RUNX2 antibody as indicated. Bar; 20 μm