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. 2007 Aug;18(8):2864–2872. doi: 10.1091/mbc.E06-12-1069

Figure 2.

Figure 2.

The human utrophin-A promoter is trans-repressed by ERF overexpression and MEK inhibition, which may be alleviated by GABPα/β. (A) Changes in subcellular fractions and subcellular localization of ERF protein in C2C12 cells caused by inhibition of MEK by using U1026. Nuclear shuttling of ERF noted over a 30-min treatment period as demonstrated by i) immunoblotting or ii) immunofluorescence by using anti-ERF antibodies, as described in Materials and Methods. Bar, 25 μm. (B) The human utrophin-A promoter is trans-repressed by ERF overexpression and MEK inhibition, which may be alleviated by GABPα/β. (i) The 1.3-kb human utrophin-A promoter-luciferase construct pPUBF showing location of the N-box/EBS according to X95523. The pPUBFΔN-box construct has a deletion that removes the N-box/EBS. (ii) pPUBF or pPUBFΔN-box derived firefly luciferase activities were normalized to pRL-TK-derived Renilla luciferase activity and expressed as a percentage of normalized luciferase activity (black column). Utrophin promoter-reporter constructs and a transfection control pRL-TK vector were cotransfected into S2 cells along with equimolar combinations of the following: GABPα/β expression vectors (GABP, gray columns), ERF expression vector pSG5-ERF (ERF, white columns), GABPα/β and ERF vectors (light gray column), or a 10 μM final concentration of MEK inhibitor UO126 (white column), and luciferase activity was assayed after a 24-h incubation. GABPα/β trans-activated the utrophin promoter construct almost fourfold (gray column), whereas decreases to 12 and 33% of normalized promoter activity was noted with ERF overexpression and MEK inhibition, respectively. However, trans-repression by ERF was completely competed by GABPα/β trans-activation (light gray column). No difference in pPUBFΔN-box reporter activity was observed upon the addition of ERF (96%). Luciferase values are the means of triplicate wells in nine separate experiments (n = 27) for pPUBF and ERF and in three separate experiments (n = 9) for MEK inhibition and GABPα/β. Error bars are SEM. (iii) Immunoblot controls showing that transfection of GABP and ERF expression vectors causes an increase in protein levels in transfected C2C12 cells.