Lentivirus production and transduction

HEK293T cells were plated on a 12-well plate, reaching 40% confluency the next day. The cells were then co-transfected with the corresponding pHR plasmid and lentiviral packaging plasmids (pMD and CMV) using Fugene HD (Promega). Cells were incubated for ~48 h and virus was collected and filtered through a 0.45-mm filter. In addition, 2 μL of polybrene and 40 μL of HEPES were added to the 2 mL viral solution. For infection, HEK293T cells were plated on a six-well plate, allowed to adhere, and reach 40% confluency. At that time, the cells were infected with 200–500 μL of viral solution. For iLID-SSPB translocation experiments, NIH3T3 cells were puromycin-selected after lentiviral transduction with an iLID-SSPB expression plasmid (pHR BFP-SSPB-SOScat-2A-PuroR-2A-iLID-CAAX) and a clonal cell line was established to limit cell-to-cell variability in expression. All imaging was done at least 48 h post infection. Cells were cultured in Dulbecco’s modified Eagle’s medium with 10% FBS, penicillin (100 U/mL), and streptomycin (0.1 mg/mL).