MK2EE Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of MK2EE
• 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 MK2EE Redistribution assay is designed as a control assay for the MK2 Redistribution assay to distinguish inhibitors acting upstream of MK2 from inhibitors specifically modulating MK2 translocation. In addition, the MK2EE assay can be used to identify compounds inhibiting nuclear export of MK2, irrespective of its activation state. In the MK2 assay, MK2 translocates from the nucleus to the cytoplasm upon p38 MAPK pathway stimulation (e.g. interleukin-1), but the two mutations (T222E and T334E) in the MK2EE assay construct result in constitutive activity and hence cytoplasmic localization of MK2EE.

Figure 1. Translocation of EGFP-MK2EE in response to Ratjadone A. Cells were treated with (right panel) or without (left panel) 50 nM Ratjadone A. Arrows indicate Ratjadone-mediated nuclear translocation detected by the image analysis algorithm.

Figure 2. Concentration-response curve of Ratjadone A in the MK2EE Redistribution assay (n=8). Concentration response was measured in 9 point half log dilution series of Ratjadone A. Cells were incubated with Ratjadone A for 60 min. Cells were then fixed and the nucleus to cytoplasm translocation was measured using image analysis. % activity was calculated relative to the positive (50 nM Ratjadone A) and negative control (0.25% DMSO).