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. 2019 Apr 25;20(8):2036. doi: 10.3390/ijms20082036

Figure 6.

Figure 6

MAPK expression and activation of TGF-β and BMP promoters in H460 cells depleted from DUSP6. (A) Protein extracts were obtained from H460ns and H460shDUSP6 cells and subjected to Western blot to determine expression levels of fibronectin (FN), ERK5, pERK5, pERK2 and α-tubulin as loading control. (B) H460ns and H460shDUSP6 cells were transfected with the corresponding reporter vectors pARE-luc and pFAST-luc as reporters for SMAD2, pCAGA-luc for SMAD3 and pBRE-luc for SMAD1/5. Protein extracts were obtained and luciferase activity was determined for each reporter. Each experiment was performed in triplicate and three different experiments were performed. Statistical significance is indicated by asterisks considering at *** for p < 0.001. (C) Western blots of phosphorylated SMAD2 and SMAD3 proteins were performed in H460ns, H460shDUSP6 and H460shDUSP6-9 cells. Numbers indicate fold activation estimated by the ratio between phosphorylated and unphosphorylated bands for each SMAD protein.