293FT cells were used to generate lentiviral particles by transfection using Lipofectamine 2000 (Life Technologies Corporation). Packaging plasmids pMD2G, PMDLg/RRE, and pRSV/Rev were cotransfected with pCDH NF1-NanoLuc C-term expression plasmid. Lentivirus containing supernatant was harvested at 48 and 72 hours after transfection. LoVo, HCT116, and HCT-15 cells were plated in respective medium with heat-inactivated FBS (10%) and 2 mM l-glutamine 2 days before infection. For infection, LoVo, HCT116, and HCT-15 cells were transduced with pCDH NF1-nanoLuc lentivirus with polybrene (8 μg/ml) for 10 hours. The cells were washed, medium was replenished, and cells were incubated for 48 hours. After this, cells were placed in puromycin selection (1 μg/ml) for 7 days. Cells were harvested for Western blots and MTT assays as described previously.

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