Fig. 6. RUNX2 ubiquitination is linked to RUNX2-S118 phosphorylation.

Western blot analysis of murine MSCs (C3H10T1/2) showing RUNX2, Bip1 protein levels after treatment with different a concentrations, b time duration of tunicamycin and c thapsigargin. d Real-time PCR analysis of RUNX2 showing increased RNA levels with tunicamycin similar to stress response genes sXBP1. e Treatment with rotenone (oxidative stress inducer) also shows similar expression of RUNX2. f Proteosomal inhibitor MG132 abrogated tunicamycin-induced loss of RUNX2 expression and association of ubiquitination. g Pre-activation of AMPK or treatment with MG132 (for 1 h) followed by tunicamycin (5 µg/ml) treatment for 12 h showing AMPK activation inhibits the effects of tunicamycin similar to MG132. h Western blot analysis showing transiently transfected phosphomimetic mutant RUNX2-S118D is resistant to stress-induced ubiquitination in HEK 293T cells. Immunofluorescence analysis showing the effects of colocalization of RUNX2 with ubiquitin i in the presence of AMPK activators (metformin and AICAR) and compound C and j tunicamycin with and without MG132. Mean ± S.E.M.; n = 3, *p < 0.1 versus untreated control; ***p < 0.001 versus untreated control