PDK1 and MRCKα colocalize in nascent lamellipodia upon EGF stimulation. MCF10A cells were cotransfected with plasmids coding for LifeAct-mTurquoise, mCherry-MRCKα, and GFP-PDK1_WT, 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 Leica AM TIRF MC equipped with a 63× oil immersion objective lens, HCX Plan-Apochromat 63×/1.47 oil CORR TIRF), 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 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 the Imaris 6.3 (Bitplane) software colocalization module and visualized with a false color LUT. Values of intensity corresponding to colors are shown in Fig. 6 A. Enlargements are obtained by bicubic 4 × 4 interpolation of the original images.
