ERF1 U2OS Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of ERF1
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

The Ets2 Repressor Factor (ERF/ERF1) is a transcriptional repressor and member of the Ets family of transcription factors. ERF1 antagonizes the activity of other Ets family members that function as transcriptional activators. The activity of ERF1 is regulated by the Ras/MEK/Erk signaling pathway that links extracellular signals with cell proliferation and differentiation. Erk-dependent phosphorylation of ERF1 controls the activity of ERF1 by modulating its cellular localization. Thus, upon mitogenic stimulation ERF1 is immediately phosphorylated by Erk and exported to the cytoplasm. Conversely, upon serum withdrawal, ERF1 is rapidly dephosphorylated and imported into the nucleus. ERF1 nuclear export is a CRM-1 dependent process and is blocked by general inhibitors of nuclear export such as Leptomycin B and Ratjadone A. The ERF1 Redistribution assay is designed to assay for inducers of ERF1 translocation by monitoring the translocation of an EGFP-ERF1 fusion protein from the cytoplasm to the nucleus in human osteosarcoma U2OS cells. Inhibition of the Ras/Erk signaling pathway by the MEK inhibitor U0126 [4-6] prevents phosphorylation and results in nuclear localisation of ERF1.

Figure 1. Translocation of EGFP-ERF1 in response to U0126. Cells were treated with vehicle control (left panel) or 10 μM U0126 (right panel). Arrows indicate U0126 mediated nuclear translocation detected by the image analysis.

Figure 2. Concentration response curves in the ERF1 Redistribution assay. Concentration response was measured in 9 point half log dilution series. Cells were incubated with test compound for 1 hour and fixed before image analysis to detect cytoplasm to nucleus translocation. A) U0126 concentration response curve in the ERF1 Redistribution assay (n=8). The EC₅₀ of U0126 is approximately 1.5 μM. B) BAY-43-9006 concentration response curve in the ERF1 Redistribution assay (n= 3). The EC50 of BAY-43-9006 is approximately 3 μM. % activity was calculated relative to the positive (10 μM U0126) and negative control (0.25% DMSO).