HCC cells (Huh7) were purchased from the Korean Cell Line Bank (KCLB; Seoul, Republic of Korea). Huh7 cells were grown in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) comprising 10% FBS (Hyclone, Thermo Fisher Scientific) and 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific) and cultured in a 5% CO2 atmosphere at 37°C. Human NIS (hNIS) gene was obtained from Dr JK Chung (Seoul National University) and the pEGFP–NI vector was purchased from Clontech (Mountain view, CA, USA). The downstream region of cytomegalovirus (CMV) promoter included the hNIS and EGFP genes, internal ribosomal entry site (IRES) was used to allow co-expression of each gene under the control of the CMV promoter. For transfection, the Huh7 cells were seeded and cultured in a 5% CO2 atmosphere at 37°C for 24 hours. When cell confluency reached 60%–70%, the purified-recombinant plasmid (containing hNIS and EGFP) was transfected by using Lipofectamine plus reagent (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s guidelines. The stable cell line was established as (Huh7/NIS) expressing hNIS, and EGFP using flow cytometry (FACSorter, BD Biosciences, San Jose, CA, USA).
Pegfp ni vector
Manufactured by Takara Bio
Sourced in United States
The PEGFP-NI vector is a circular plasmid DNA that can be used to express enhanced green fluorescent protein (EGFP) in mammalian cells. It contains the EGFP gene under the control of the human cytomegalovirus (CMV) immediate-early promoter.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipofectamine plus reagent, by Thermo Fisher Scientific (1 mentions)
Facsorter, by BD (1 mentions)
Lipofectamine ltx and plus reagent, by Thermo Fisher Scientific (1 mentions)
Prl tk renilla reporter vector, by Promega (1 mentions)
Dmlfsa, by Leica (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
4 protocols using pegfp ni vector
1
Establishment of Huh7/NIS Stable Cell Line
2
Murine Spindle Cell and Keratinocyte Transfection
3
Galr1-EGFP Fusion Protein Expression
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Gal1R-EGFP vectors were made by reverse transcribing the open reading frame of the rat Galr1 gene, followed by PCR amplification using the primers as shown in SI Table 3 . PCR products were ligated into a pEGFP-NI vector (Clontech) at its HindIII/SacII and Hind/KpnI restriction sites to generate Galr1-EGFP expression vectors. Rat pheochromocytoma cells (PC12) (1,000,000 cells/plate) were transfected with Galr1-Egfp (1 µg plasmid DNA) using Effectene Transfection Reagent (Qiagen), as described earlier46 (link). Stably transfected cells were selected by growing them in the presence of geneticin (G418, Sigma) at a concentration of 800 µg/ml. PC12 cells were sub-cultured and used for 10–15 passages. For Gal1R-EGFP internalization, cells were seeded on 13-mm glass coverslips (10–20,000 cells/well in 24-well plates) previously coated with poly-D-lysine (Sigma), and pharmacologically probed 72 h after plating. Galanin was superfused at a concentration of 100 nM. Cells were photographed using differential interference contrast (DIC) microscopy (DMLFSA, Leica).
4
Characterization of NMNAT2 and Palmitoyl Transferases
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Mouse zDHHC-HA constructs were a gift from Luke Chamberlain (Glasgow, UK) with kind permission from Masaki Fukata (Okazaki, Japan). Note that throughout this paper, palmitoyltransferases are referred to according to the new standard zDHHC nomenclature. For creation of EGFP-tagged zDHHC constructs, relevant coding sequences were transferred to the pEGFP-NI vector (Clontech). Fusions of wild-type and variant Nmnat2 with FLAG, EGFP, and photoactivatable GFP (PA-GFP) tags (11 (link)) as well as FLAG-WldS and FLAG-Nmnat1 (1 (link)) were described previously. Note that cISTID-PA-GFP and FLAG-Nmnat2ΔcISTID constructs were previously referred to as Exon6-PA-GFP and FLAG-Nmnat2Δex6, respectively (11 (link)). Table 1 provides an overview of all NMNAT2 mutants used in this study. FLAG-APT1 and FLAG-APT2 constructs were generated by PCR amplification of the APT1 (NM_008866.2) and APT2 (NM_011942.1) coding sequences from a mouse cDNA library and insertion into pCMV-Tag2A vector (Stratagene). Enzymatically inactive zDHHC7 (C160S referred to as zDHHS7), zDHHC17 (C467S; zDHHS17), APT1 (S119A), and APT2 (S122A) constructs were created using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.
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