PDK1 and MRCKα colocalize to the cell membrane upon EGF stimulation. (A) MCF10A cells were transfected with GFP-PDK1_WT construct, then plasma membrane–bound PDK1 was detected through TIRF microscopy. Cells were stimulated with 5 ng/ml EGF or not stimulated, and imaged every 20 s. (A, left) TIRF fluorescence images of representative cells before and after EGF stimulation. (A, right) Time-dependent quantification of mean fluorescence intensity of the indicated number of experiments. Error bars represent the standard error of the mean. (B) MCF10A cells were transfected with GFP fused constructs of PDK1_WT, PDK1_L155E, PDK1_K465E, and PDK1_KD, then assayed as in A. (C) MCF10A cells expressing mCherry-MRCKα were assayed as in A. (D) MCF10A coexpressing GFP-PDK1_WT and mCherry-MRCKα were stimulated with 5 ng/ml EGF, and the colocalization channel was calculated as described in the Materials and methods section. In the colocalization channel, cell shapes are outlined in white. (E) EGF induced time-dependent variation of mCherry-MRCKα colocalization with GFP-PDK1_WT, GFP-PDK1_L155E, GFP-PDK1_K465E, or GFP-PDK1_KD. Error bars represent the standard error of the mean of n independent experiments. Bars, 20 µm.