Short hairpin sequences in pLKO.1-puromycin lentiviral vector (Sigma-Aldrich) were used to generate stable KD. The specific shRNA sequences used are shown in Table S2 . GFP-tagged ARFs were kind gifts from P. Melançon (University of Alberta, Edmonton, Canada) and alternate fluorescent tags, mutations, and chimeras were generated by sub-cloning. All RNAi-resistant variants and chimeras were made by mutagenesis or sub-cloning using fragment synthesis (GeneArt). Plasmids will be deposited with Addgene upon publication.
Stable cell lines were made by co-transfecting lentiviral packaging vectors VSVG (pMD2.G; plasmid 12259; Addgene) and psPAX2 (plasmid 12260; Addgene) with plasmid of interest, into HEK293-FT using Lipofectamine 2000 (Thermo Fisher Scientific) as per manufacturer’s instructions. Viral supernatants were filtered using PES 0.45 μm syringe filters (Starlab) and concentrated using Lenti-X Concentrator (Clontech) as per the manufacturer’s instructions. PC3 cells were transduced with lentivirus for at least 3 d prior to FACS sorting or selection with 300 μg/ml G418, 2.5 μg/ml puromycin (both Thermo Fisher Scientific) or 10 μg/ml blasticidin (InvivoGen). To allow direct comparison PC3 cells expressing mNG were used for shRNA expression and Scr shRNA was added to cells over-expressing mNG-tagged plasmids.
Stable cell lines were made by co-transfecting lentiviral packaging vectors VSVG (pMD2.G; plasmid 12259; Addgene) and psPAX2 (plasmid 12260; Addgene) with plasmid of interest, into HEK293-FT using Lipofectamine 2000 (Thermo Fisher Scientific) as per manufacturer’s instructions. Viral supernatants were filtered using PES 0.45 μm syringe filters (Starlab) and concentrated using Lenti-X Concentrator (Clontech) as per the manufacturer’s instructions. PC3 cells were transduced with lentivirus for at least 3 d prior to FACS sorting or selection with 300 μg/ml G418, 2.5 μg/ml puromycin (both Thermo Fisher Scientific) or 10 μg/ml blasticidin (InvivoGen). To allow direct comparison PC3 cells expressing mNG were used for shRNA expression and Scr shRNA was added to cells over-expressing mNG-tagged plasmids.