Fig 2: Characterization of the Rictor binding domain in Pdcd4. A, Pdcd4(1-156) binds with Rictor. HEK293 cells were transfected with HA-tagged full-length (WT), Pdcd4(1-156) and Pdcd4(157-469) plasmids and immunoprecipitated with an anti-HA antibody. B and C, Pdcd4(1-156) inhibits mTORC2 kinase activity. B, representative images of mTORC2 kinase assay; C, quantification of mTORC2 kinase activity. The ratio of p-Akt/precipitated mTOR in control cells was set to 100%. Data were analyzed by a one-sample t-test (n=3; mean±s.d.; **p < 0.01). D, Deletion analyses to map the Rictor binding domain. HA-tagged full-length (WT) and various Pdcd4 deletion mutant plasmids were transfected into HEK293 cells and immunoprecipitated with an anti-HA antibody. E, Amino acid sequence of RBD. The charged amino acid residues are in red. F, Site-specific mutagenesis analysis to identify critical residues for Rictor binding. Various HA-tagged Pdcd4 mutant plasmids were transfected into HEK293 cells and immunoprecipitated with an anti-HA antibody. G, Pdcd4(1-156)(3A) does not bind with Rictor. HA-tagged Pdcd4(1-156) and Pdcd4(1-156)(3A) plasmids were transfected into H1299 cells and immunoprecipitated with anti-HA antibody. H, RBD but not RBD(3A) peptide binds with Rictor. HA-tagged RBD and RBD(3A) peptides were incubated with H1299 cell lysate and immunoprecipitated with an anti-HA antibody. I, Methylated RBD peptide does not bind with Rictor. HA-tagged RBD and methylated RBD (Me-RBD) peptides were incubated with H1299 cell lysate and immunoprecipitated using an anti-HA antibody. All immunoprecipitation assays were followed by Western blot analysis with the indicated antibody.