Pvsv g vector

The methods used in the present study have been previously described [24 (link)]. Briefly, pBABE with a full-length wild-type EGFR cDNA fragment was purchased from Addgene (Cambridge, MA). pBABE constructs encoding the EGFR L858R mutation and the EGFR L858R mutation plus each of the resistant mutations (L858R + L747S, L858R + D761Y, L858R + T854A, and L858R + T790 M) were generated using the PrimeSTAR Mutagenesis Basal Kit (TaKaRa, Otsu, Japan). All primer sequences are available upon request. All the mutations were confirmed using direct sequencing experiments. The pBABE constructs were cotransfected with a pVSV-G vector (Clontech, Mountain View, CA) to generate the viral envelope in gpIRES-293 cells using the FuGENE6 transfection reagent (Roche Diagnostics, Basel, Switzerland) to produce viral particles. After 48 h of transfection, the culture medium was collected and the viral particles were concentrated by centrifugation at 15,000 ×g for 3 h at 4 °C. The viral pellet was then resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, St. Louis, MO) and was added to Ba/F3 cells. Infected Ba/F3cells were then purified using GFP-based fluorescence-activated cell sorting using the BD FACS Aria Cell Sorter Special Order Research Product (BD Biosciences, Franklin Lakes, NJ).

To generate stable Girdin knockdown 293FT cells, 24 μg of either control or Girdin shRNA and 4 μg of vesicular stomatitis virus G protein (pVSV-G) vector (Clontech) were cotransfected into GP2-293 packaging cells (Clontech Laboratories). Twenty-four hours after the transfection, the medium was replaced with 5 mL of fresh DMEM containing 10% FBS and the cells were further cultured at 32°C to facilitate virus production. HeLa cells were infected with virus-containing supernatants harvested 48 h post-transfection, followed by selection with 2 μg/mL puromycin.