HEK293, 293 T and DLD1 cells were obtained from American Type Culture Collection (ATCC), DLD1-PDK1−/− and counterpart cells were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University School of Medicine), and these cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. Cell transfection was performed using Lipofectamine and Plus reagents, as described previously44 (link). Packaging of lentiviral shRNA or cDNA expressing viruses and retroviral cDNA expressing viruses, as well as subsequent infection of various cell lines were performed according to the protocols described previously44 (link). Following viral infection, cells were maintained in the presence of puromycin (0.5 μg/ml).
Cell fractionations were performed with Cell Fractionation Kit (CST9038). Kinase inhibitors Mk2206 (Selleck S1078), Rapamycin (Selleck S1039), PP242 (Selleck S2218) and PF-4708671 (Selleck S2163) were used at the indicated doses. Growth factors including EGF (Sigma E9644) and insulin (Invitrogen 41400-045), were used at the indicated doses. PIP3 beads (P-B00Ss) and label-free PIP3 (P-3908) were purchased from Echelon Biosciences.
Cell fractionations were performed with Cell Fractionation Kit (CST9038). Kinase inhibitors Mk2206 (Selleck S1078), Rapamycin (Selleck S1039), PP242 (Selleck S2218) and PF-4708671 (Selleck S2163) were used at the indicated doses. Growth factors including EGF (Sigma E9644) and insulin (Invitrogen 41400-045), were used at the indicated doses. PIP3 beads (P-B00Ss) and label-free PIP3 (P-3908) were purchased from Echelon Biosciences.
Free full text: Click here
