Pcmv sport6 vector

A pCMV-SPORT6 vector containing the NLRC4 cDNA MGC: 35330 was obtained from Thermo Scientific. Subsequent cloning was performed by Bioinnovatise, Inc (Rockville, MD). Briefly, after DpnI digestion, the A to T mutation at position 1009 of NLRC4 was introduced into this plasmid using site-directed mutagenesis and complementary primers (see Supplementary Table 3) containing the patient’s mutation. An IRES-eGFP sequence was then cloned downstream of the WT and mutant NLRC4 open reading frames by seamless cloning. Finally, a 24 bp c-terminal FLAG tag was appended to each construct to generate pCMV-Sport6-NLRC4flag-GFP and pCMV-Sport6-T337SNLRC4flag-GFP. PCR fragments containing either WT or T337S mutant NLRC4flag were inserted into the multiple cloning site of the migR1 vector^{42 (link)} by seamless cloning to generate NLRC4flag-MigR1 and T337SNLRC4flag-MigR1. All constructs were completely sequenced to confirm position.

The following plasmids were used. pcDNA3.1, pcDNA3.1-2xFLAG-SREBP1c (Addgene plasmid 26802)^{18 (link)} and pShuttle-CMV (Addgene plasmid 16403)^{19 (link)}. A pCMV-Sport6 vector encoding the mouse Nrf1 cDNA (GenBank accession: BC047283.1) was acquired from Open Biosystems (ThermoScientific). The Nrf1 cDNA was PCR amplified with the addition of tag elements incorporated onto its 5′ (his) or 3′ (flag or myc) ends, along with suitable restriction sites, which were then utilized to subclone into the pShuttle-CMV vector.

The full length cDNA of human FoxO1, wild type or the mutant, were subcloned into the pCMV Sport6 vector (Life Technologies), and the reporter vector pGL3-G6PC1 containing the region of the human G6PC1 promoter encompassing the dominant region B was constructed and used for luciferase assays. HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin G, 50 μg/ml streptomycin (Sigma), and 0.1 mM non-essential amino acids (Invitrogen). HeLa cells were transfected using Opti-MEM and recommendations. LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer’s Briefly, a total of 30 ng of pCMV FoxO1 and 50 ng of pGL3-G6PC1 and 10 ng of pRL-TK (control renilla luciferase vector) were used for transfection of 1 × 10⁵ cells seeded on a 24-well plate one day before transfection. After transfection and incubation, cells were washed with 1X PBS and lysed with luciferase lysis buffer supplied with the Luciferase assay kit (Promega). Luciferase activity was measured using the Dual Luciferase assay system (Promega) and Lmax Luminometer (Molecular Devices). All values were normalized by the relative ratio of firefly luciferase activity and renilla luciferase activity. At least four independent transfections were performed in duplicate.