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. 2010 Mar 26;5(3):e9910. doi: 10.1371/journal.pone.0009910

Figure 1. AKT1 interaction with PDK1 stabilized by the reconstituted IFP.

Figure 1

(A) Schematic diagram showing the split IFP reconstitution mediated by the interaction between AKT1 and PDK1. IFPN was fused to the N-terminus of AKT1. IFPC was fused to the C-terminus of PDK1. Neither fusion protein was fluorescent. The interaction between AKT1 and PDK1 brings IFPN and IFPC in proximity and reconstitutes IFP fluorescence. The reconstituted IFP stabilizes the PDK1-IFPC::IFPN-AKT1 complex. (B) Association of PDK1-IFPC with IFPN-AKT1. HeLa cells stably expressing the PDK1-IFPC::IFPN-AKT1 complex were lysed in NP40 lysis buffer. Immunoprecipitation was performed with the indicated antibodies (top labels), followed by western blotting with anti-PDK1 (upper blot) and anti-AKT (lower blot). Lane 1, IP with normal IgG. Lane 2, IP with anti-AKT1. Lane 3, blank. Lane 4, total cell lysate. (C) Translocation of the PDK1-IFPC::IFPN-AKT1 complex upon PI3K signaling activation or inhibition. Cells stably expressing the PDK1-IFPC::IFPN-AKT1 complex were serum starved for overnight then treated with LY294002 (20 µM) for 3 hours (image 2), 10% serum for 10 minutes (image 3), and LY294002 (20 µM) for 3 hours and then 10% serum for 10 minutes (image 4). At least 100 cells were examined from different fields for each sample with 100% of the examined cells in each sample showing the same fluorescence localization presented. (D) Accumulation of the PDK1-IFPC::IFPN-AKT1 complex in nucleus with LMB treatment. HeLa cells stably expressing the PDK1-IFPC::IFPN-AKT1 complex were starved overnight and then treated with LMB at 50 nM for 3 hours. Nuclei were stained with Hoechst 33342.