Plasmid construction. To construct pFT-GFP-CFTR△F508 for stably expressing GFP-CFTR△F508 in the Flp-In T-REx system (Invitrogen, Carlsbad, CA),
VSV-G tag cDNA fragment encoding GFP-CFTR△F508 [14] was inserted into pcDNA5/FRT/TO (Invitrogen). pFT103 was constructed by inserting a fragment encoding One-STrEP-tag (a modified Strep-tagII), which was excised from pEXPR-IBA103 (IBA, St. Louis, MO), into the multiple cloning site of pcDNA5/FRT/TO. Full-length cDNA for rat and human UBXD1 was amplified respectively from a rat liver and a human kidney cDNA library (Clontech, Palo Alto, CA) by PCR. The rat UBXD1 cDNA was subcloned into pFLAG-CMV-6 (Sigma–Aldrich, St. Louis, MO) to express FLAG-tagged UBXD1, and into pcDNA5/FRT/TO (Invitrogen) to express UBXD1 in the doxycycline (a tetracycline derivative) inducible Flp-In T-REx system. The human UBXD1 cDNA was subcloned into pGEX-6P-1 (GE Healthcare, Piscataway, NJ) to express UBXD1 as a glutathione-S-transferase (GST)-fused protein, and into pFT103 to express strep-tagged UBXD1. Expression
plasmids for the GST- or FLAG-tagged SVIP, Ufd1, and p47 were described previously [5].
