Monitoring DYRK1A and NFATc1 Interaction in HEK293T Cells

Based on the procedure detailed in Grygier et al.^{34 (link)}, the experiment was conducted as follows: HEK293T cells were grown on μ-Slide 8 well (IBIDI) to 50–70% confluency. The plasmids expressing the desired proteins, mCherry-DYRK1A and eGFP-NFATc1, were transiently co-transfected with PEI Prime (Sigma-Aldrich). 24 h later cells were pre-treated with compounds (10 μM) or DMSO for 3 h and then stimulated with IM (5 μM) (Thermo Fisher Scientific) for 1 h. Cells were washed with 1 ml PBS and the nuclei were stained with Hoechst 33258 (ThermoScientific) for 10 min at room temperature and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. Images were collected with Zeiss Axio Observer 3 fluorescence microscope with 40 × objective and analysed in ZEN Blue edition software.