PTD-A2, an AMIGO2 competitive peptide, blocks PDK1 translocation and Akt activation. (A) The TAT-A2 binding assay was performed. PH domain proteins (0–60 µg) were incubated with 1-µM FITC–TAT-A2 and detected by fluorescence. Data were collected from independent experiments and analyzed using a two-tailed unpaired t test. n = 4. (B) Peptide competition assay with Con or TAT-A2 and CD to PH domains. His-tagged PDK1^PH was incubated with purified GST-AMIGO2^CD and Con or TAT-A2 in a dose-dependent manner. His-tagged PH domain proteins were pulled down with GST resins, eluted, and analyzed by Western blotting. (C) Con- and TAT-A2– treated HUVECs were immunoprecipitated with an AMIGO2 antibody and blotted with anti-PDK1 and AMIGO2 antibodies. (D) Localization of PDK1 in the plasma membrane and cytosol of HUVECs in the presence of VEGF after Con or TAT-A2 treatment. Bars, 20 µm. (E) Subcellular localization analysis was performed by cell fractionation in VEGF-treated, TAT-A2–treated HUVECs. (F) Quantification of Western blots. Subcellular localization analysis was performed by cell fractionation with or without VEGF in control and TAT-A2 HUVECs. HUVECs were affected by both PTD-A2 peptides. (G) Effects of TAT-A2 on VEGF-induced Akt signaling in HUVECs. Con, control peptide; TAT-A2, TAT-RVVFLEPLKD peptide. PTD-A2 means both TAT-A2 and NGR-A2. Data are means ± SD. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. HC, heavy chain; IB, immunoblotting; IP, immunoprecipitation; WCL, whole cell lysate.