Pcdna3.1 myc his expression vector

Recombinant DNA techniques were performed according to standard procedures (Sambrook et al., 1989 ) and the integrity of all constructed plasmids was confirmed by extensive sequencing analyses. Site-directed mutagenesis was carried out using DpnI. A p3xFlag-CMV-14 expression vector (Sigma) and pcDNA3.1-MycHis expression vector (ThermoFisher) were used to express a protein (EDEM2 or TXNDC11) tagged with 3xFlag and c-Myc at the C-terminus, respectively. pCMV-SP-TAP-EDEM2 was constructed based on pCMV, pcDNA3.1-SP-2xProA-2xTEV-6xMyc and pCMV-EDEM2-3xFlag using an NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs). SP denotes a signal peptide. The ERAD-Ls substrate mCD3-δ-ΔTM-HA was the kind gift of Maurizio Molinari at the Institute for Research in Biomedicine, Switzerland.

HSP60 cDNA was purchased from the Korea Human Gene Bank (KRIBB, Daejon, Korea) and cloned into the HindIII/XbaI sites of pcDNA3.1(+)-Myc-His expression vector (Thermo Fisher Scientific). 293FT (3~4×10⁵ cells/well) cells were transfected with pcDNA3.1(+) or pcDNA3.1(+)-HSP60-myc-His vectors, using Lipofectamine™ 2000 (Thermo Fischer Scientific) according to the supplier’s protocol. Protein was transiently expressed for 48 h, and the cell pellet was lysed in 1% NP40 lysis buffer. Cell lysates were subjected to Western blot analysis or immunoprecipitation with anti-Myc (Thermo Fisher Scientific), anti-His (Thermo Fisher Scientific), or anti-HSP60 antibodies (Santa Cruz Biotechnologies). To knock down HSP60, H9 cells pre-incubated with 10 μm Y-27632 for 1 h were dissociated with TrypLE Select (Thermo Fisher Scientific), and 2×10⁵ cells were seeded into Matrigel (BD Biosciences) coated-feeder free plate in TeSR-E8 medium (STEMCELL Technologies, Vancouver, Canada). The next day, the cells were transfected with HSP60 siRNA #1 and #2 (Bioneer, Daejeon, Korea) or accutarget negative control siRNA (Bioneer) using RNAimax (Thermo Fisher Scientific) according to the supplier’s protocol. The sense sequences of HSP60 siRNA #1, 2 were 5′-GUGUUGAAGGAUCUUUGAU-3′ and 5′-GAAGUUUGAUCGAGGCUAU-3′, respectively. After transfection, the cells were incubated for 48 h before further analysis.

The human FasL luciferase reporter construct (HFLP-Luc) containing 1.2 kb of the FASLG promoter, and the empty control plasmid HsLuc have been described previously.^{38 (link)} HFLP-Luc with single or double mutated LRH-1 binding sites in the FASLG promoter (HFLP mut BS1, HFLP mut BS2, HFLP mut BS1+2) were generated by site-directed mutagenesis using a kit from Stratagene (Quick-Change, Agilent) with primers shown in Table 1. The putative LRH-1 binding sites and in the FASLG promoter and their mutation are depicted in Figure 2a.
The LRH-1 reporter containing 5 copies of the LRH-1 binding motive of the human SHP promoter in the pGL3 basic plasmid (Promega, Mannheim, Germany) has been described previously.^{39 (link)} The Myc/6xHis-tagged LRH-1 expression plasmid was generated by cloning human LRH-1 into a pcDNA3.1 Myc/His expression vector (Invitrogen). The FLAG-tagged LRH-1 expression plasmid was generated by exchanging the Myc/His tag with a 3xFLAG epitope tag. A β-galactosidase expression plasmid was used to normalize transfection efficiency (Invitrogen).