EGF-induced PDK1-MRCKα colocalization at the plasma membrane requires PDK1 PIF-binding pocket and binding to PIP3. MCF10A were cotransfected with plasmids coding for mCherry-MRCKα (red) and GFP-PDK1_WT, GFP-PDK1_L155E, GFP-PDK1_K465E, or GFP-PDK1_KD (green), 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 parts of the 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 Imaris 6.3 (Bitplane) software and visualized with a false color LUT. Values of intensity corresponding to colors are shown in Fig. 6 A.
