PDK1 interacts with MRCKα through the PIF-binding pocket. (A) HeLa cells were stably transduced with lentivirus coding for PDK1_WT, PDK1_L155E, or an empty vector and then transfected with a plasmid driving Flag-MRCKα expression. Immunoprecipitation was performed using an anti-flag antibody. Immunoprecipitated PDK1 was detected by Western blotting. (B) GST-fused MRCKα catalytic subunit (GST-MRCKα_CAT) was purified from 293T cells and used to pull down PDK1 from lysates of MCF10A stably transduced with lentiviral vectors coding for PDK1_WT, PDK1_L155E, PDK1_K465E, or an empty vector. Pulled-down PDK1 was detected by immunoblotting. (C) GST-PDK1_WT was produced and purified from 293T and used to pull down GFP-MRCKα_CAT. Pulled-down GFP-MRCKα_CAT was detected by immunoblotting. (D) MCF10A cells were infected with a lentivirus coding for myc-PDK1 and, after EGF deprivation, stimulated for 200 s with EGF 5 ng/ml. PLA of myc-PDK1 and MRCKα was performed using as detecting antibodies anti–c-Myc produced in mouse and anti-MRCKα produced in rabbit or with the combination of unspecific immunoglobulins (IgG M and IgG R). Cellular shape was identified by Phalloidin-488 staining. Bar, 10 µm.