Pegfp n1 backbone

The plasmid encoding GFP-ARF1 WT was obtained from Addgene (#39554; Cambridge, MA). The Q71L mutation was introduced via Quikchange mutagenesis and confirmed by sequencing (Stratagene, La Jolla, CA), using the following mutagenic primers: 5′-GACGTGGGTGGCCTGGACAAGATCCGG-3′ and its complement, 5′-CCGGATCTTGTCCAGGCCACCCACGTC-3′. The Venus-GBF1 A795E construct was previously described (Lanke et al., 2009 (link)). The A795E mutation provides resistance to BFA but otherwise this construct behaves similarly to WT (Lanke et al., 2009 (link)). The GBF1 A795E coding region was subcloned into the pEGFP N1 backbone (Clontech, Mountain View, CA). The plasmid encoding ARL1-GFP was a gift from Richard Kahn through Addgene (plasmid #67395).
Transfection was performed using the X-tremeGENE 9 DNA transfection reagent (Roche, Indianapolis, IN) in accordance with the manufacturer’s protocol. For all transfection experiments, 35-mm dishes of HeLa cells were transfected with 0.2 μg of plasmids encoding GFP-ARF1 WT, GFP-ARF1 Q71L, or ARL1-GFP, or 0.5 μg of the plasmid encoding GFP-GBF1 A795E. Experiments were performed 16-17 h posttransfection.

pFCaGW is a lentiviral vector that expresses EGFP (G) under the CaMKII promoter (Ca). The pCaMKII-EGFP transcriptional unit was assembled into a custom backbone (derived from (57 (link)), a gift from Alexandros Poulopoulos, and the pEGFP-N1 backbone (Clontech)) by Golden Gate Assembly (NEB), with the CaMKII promoter from mCh-GluA1-CIB (a gift from Matthew Kennedy (Addgene plasmid # 89444; http://n2t.net/addgene:89444; RRID:Addgene_89444)), and EGFP from pEGFP-N1. pCaMKII-EGFP was inserted by NEB HIFI Assembly into the PacI site of a modified pFUGW vector (pFW), where the ubiquitin promoter and EGFP were deleted by KLD mutagenesis (NEB), yielding pFCaGW. For all cloning reactions, fragments were generated by PCR with KAPA HiFi HotStart DNA polymerase (Roche) or by DNA synthesis (IDT). psPAX2 (Addgene plasmid # 12260; http://n2t.net/addgene:12260; RRID:Addgene_12260) and pMD2.G (Addgene plasmid # 12259; http://n2t.net/addgene:12259; RRID:Addgene_12259) were gifts from Didier Trono.

The plasmids used for expression of iRFP670 and iRFP720 in mammalian cells were based on pEGFP-N1 backbone (Clontech). The iRFP670 and iRFP720 genes were PCR-amplified as AgeI-NotI fragments and swapped with the EGFP gene in pEGFP-N1 vector, thus generating the piRFP670 and piRFP720 plasmids.
A rat adenocarcinoma MTLn3 cell line was grown in αMEM medium (Life Technologies) containing 5% fetal bovine serum, 0.5% penicillin-streptomycin, and 2 mM glutamine. Plasmid transfections were performed using an Effectene reagent (Qiagen) according to the manufacturer's protocol. Stably expressing preclonal cell mixtures were selected with 700 μg/ml G418 antibiotic. Sorting of fluorescent cells was performed using a MoFlo XDP sorter (Beckman Coulter) equipped with a 676 nm Kr laser and a 700 nm long-pass emission filter.