Generation of Myristoylated-Akt1 and IκB-SS Lentiviruses

Myristoylated-Akt1 (myrAkt1) lentivirus was generated by co-transfecting pCCL-myrAkt1 backbone^{15 (link)} (13 μg) with RRE (5 μg), REV (2.5 μg), and VSV-G (3 μg) packaging plasmids on a 10 cm dish of 80% confluent 293T/17 cells (ATCC CRL-11268) using Lipofectamine 2000 (ThermoFisher Scientific 12566-014) according to the manufacturer’s suggestions. Forty-eight hours post-transfection, supernatants were processed using a Lenti-X Concentrator (ClonTech 631232) according to the manufacturer’s suggestions. Precipitated myrAkt1 lentivirus was resuspended in 0.5 mL TNE Buffer (50 mM Tris pH 8.0, 1 mM EDTA, 130 mM NaCl), aliquoted, and stored at −80 °C. Viral titers were determined using Lenti-X p24 Rapid Titer Kit (ClonTech 632200). IkB-SS expressing lentiviral vector was generated by sub-cloning the sequence of human IκBα super-suppressor (Addgene #15264) into pLVX Puro Vector (Clontech #632164). IkB-SS lentivirus was generated by co-transfecting pLVX-Puro-IkB-SS vector with RRE, REV, and VSV-G packaging plasmids in 293T cells as described earlier. pLVX Puro Vector (Clontech #632164) was utilized to generate the “Puro-empty” lentivirus.