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Fig. 10. Inhibition of MKP-1 attenuates the DEX-mediated decrease in Runx2 serine phosphorylation. Primary dermal fibroblasts were transduced with Runx2 retrovirus or left unmodified for controls and cultured in osteogenic media with (+) and without (–) 10 nM DEX. After 7 days in culture, cells were treated with vehicle (ethanol), vehicle+DEX (10 nM), sanguinarine (50 µM), and sanguinarine (50 µM)+DEX (10 nM) for 30 minutes. (A) Western blot analysis of whole-cell lysates after 7 days in culture was conducted with antibodies against MKP-1, MKP-3, phospho-ERK (phospho-p44 ERK, phospho-p42 ERK) and ERK (p44 ERK and p42 ERK). GAPDH was used as a loading control. Blot are representative of data from three separate experiments in triplicate. (B) Runx2 phosphoserine levels were assessed by immunoprecipitation of whole-cell lysates with an antibody against Runx2 and western blotting with antibodies against Runx2 and phosphoserine (pSerine). Blots are representative of data from two separate experiments in triplicate.
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