Lentiviral Transduction of Cell Lines

cDNA for pLenti CMV GFP Puro (658-5) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid #17448^{76 (link)}). Portions of paxillin-pEGFP (a gift from Rick Horwitz, Addgene plasmid # 15233) or mRuby-Paxillin-22 (a gift from Michael Davidson, Addgene plasmid #55877) were subcloned into a modified version of the pRRLSIN.cPPT.PGK-GFP.WPRE (a gift from Didier Trono, Addgene plasmid # 12252) by restriction enzyme cloning. pLenti.PGK.LifeAct-GFP.W was a gift from Rusty Lansford (Addgene plasmid # 51010). To generate lentivirus, plasmids were co-transfected with pCMV-VSVG (a gift from Bob Weinberg, Addgene plasmid #8454), pMDLg/pRRE, and pRSV-REV (gifts from Didier Trono, Addgene plasmid #12251 and #12253^{77 (link),78 (link)}) in 293T cells using the calcium phosphate precipitation method⁷⁹ . Viral supernatants were collected after 48 h, concentrated with PEG-it^TM (System Biosciences, Palo Alto, CA) following the manufacturer’s protocol, filtered through a 0.45 μm filter (ThermoFisher Scientific Nalgene, Waltham, MA), and stored at −80 °C. Viral titer was determined by serial dilution and infection of NIH3T3s in the presence of 10 μg mL⁻¹ polybrene (Santa Cruz Biotechnology, Dallas, TX). Titers yielding maximal expression without cell death or detectable impact on cell proliferation or morphology were selected for studies.