Construction of FLAG-Tagged Fusion Proteins

All primers used in these cloning projects are listed in Table 1 (Supplementary Material). To generate amino-terminal 3XFLAG fusion constructs, PCR was used to introduce appropriate 5′ and 3′ restriction sites to furin, syntaxin 4, and Tgn38 cDNA (Origene, Rockville, MD). The PCR products were cloned in frame to the 3XFLAG coding sequence in the p3XFLAG-CMV 7.1 expression vector (Sigma Aldrich, St. Louis, MO). The GeneTailor Site-Directed Mutagenesis system (Life Technologies) was used to create 3XFLAG-syntaxin 4+YGRL, 3XFLAG-syntaxin 4 +YKGL, and 3XFLAG-syntaxin 4 +YQRL and 3XFLAG-TGN38 _YGRL, 3XFLAG-TGN38ΔYQRL, and 3XFLAG-furinΔYKGL constructs using previously described conditions (Moore et al., 2011 (link)). The 3XFLAG-furin_YGRL mutant was constructed by three-step site-specific PCR mutagenesis (Shokeen et al., 2008 (link)) using end, mutagenic and overlapping primers listed in Table 1. The overlap extension PCR cloning method (Bryksin and Matsumura, 2010 (link)) was used to create the 3XFLAG-syntaxin 6 and 3XFLAG-syntaxin 6 ΔYGRL (both constructs previously described in Moore et al., 2011 (link)) into the pcDNA3.1mycBioID plasmid (plasmid # 35700, Addgene.org) (Roux et al., 2012 (link)). All constructs were confirmed by sequencing (Eurofins MWG Operon, Hunstville, AL).