EGF induces an increase of PDK1 and MRCKα colocalization at the plasma membrane. MCF10A were cotransfected with GFP-PDK1_WT (green) and mCherry-MRCKα (red) plasmids, deprived of EGF for 6 h, and kept in a humidified chamber at 37°C and 5% CO2. Cells were then imaged by means of TIRF microscopy (True MultiColor Laser TIRF AM TIRF MC equipped with a 63× oil immersion objective lens, HCX Plan-Apochromat 63×/1.47 oil CORR TIRF; Leica), which allowed us to image the part of cells close to the coverslip. The depth of the evanescent field was kept at 90 nm. Cells were imaged over a time period of 660 s and stimulated or not stimulated with 5 ng/ml EGF. The time at which the stimulus was added was set to t = 0 s. Images were acquired every 20 s. Colocalization channel was calculated by Imaris 6.3 (Bitplane) software and visualized with a false color LUT. Values of intensity corresponding to colors are shown in Fig. 6 A.
