Generating PH_AKT-TagRFP-T Plasmids

To generate PH_AKT–TagRFP-T plasmids, DNA sequences for wildtype and R25C mutant of PH_AKT from PH-Akt-GFP plasmid (#51465, Addgene) and PH-Akt(R25C)-GFP plasmid (#51466, Addgene) were amplified by PCR and cloned into pmTagRFP-T-N1 (50 (link)) using XhoI and BamHI sites. PH_AKT–stagRFP was generated by site-directed mutagenesis of TagRFP-T (D159V) in the PH_AKT–TagRFP-T plasmid. Exo70(K632A, K635A)-CRY2-mCherry, CIB1-CAAX, and TfRc-pH were previously described (16 (link)). All cloning was done using standard molecular biology techniques. Es2 was purchased from Sigma or Cayman Chemical and stored as a 200-mM stock solution in DMSO at −30°C. Wortmannin, gefitinib, and afatinib were purchased from Tocris Bioscience and stored as 10 mM, 100 mM, and 20 mM stock solutions, respectively, in DMSO at −30°C.