To express the components of the NLRP3-inflammasome (NLFP3-I), we utilized four plasmids expressing mouse NLRP3 (pcDNA3-N-Flag-NLRP3, Addgene plasmid # 75127), ASC (pcDNA3-N-Flag-ASC1, Addgene plasmid # 75134), CASP1 (pcDNA3-N-Flag-Caspase-1, Addgene plasmid # 75128) and pro-IL-1B (pCMV-pro-Il1b-C-Flag, Addgene plasmid # 75131). The use and construction of the NLRP3-I expression plasmids were previously described (25 (link)).
The plasmid vector used to express mitochondrial-targeted catalase (mCAT) and its respective control vector were p-mCAT (VectorBuilder ID VB170403-1078nzg) and p-EV (VectorBuilder ID VB210726-1273jte). The p-mCAT transgene cassette was transcribed from the 212 nucleotide elongation factor α1 “short” (EFS) promoter. The EFS promoter transcribes a polycistronic transcript encoding EGFP (enhanced green fluorescent protein), a self-cleaving 2A peptide site, followed by mCAT cDNA, and terminated by a simian virus 40 (SV40) late polyA sequence. p-EV is identical to the p-mCAT construct, except lacking the EGFP and mCAT sequences.
The plasmid vector used to express mitochondrial-targeted catalase (mCAT) and its respective control vector were p-mCAT (VectorBuilder ID VB170403-1078nzg) and p-EV (VectorBuilder ID VB210726-1273jte). The p-mCAT transgene cassette was transcribed from the 212 nucleotide elongation factor α1 “short” (EFS) promoter. The EFS promoter transcribes a polycistronic transcript encoding EGFP (enhanced green fluorescent protein), a self-cleaving 2A peptide site, followed by mCAT cDNA, and terminated by a simian virus 40 (SV40) late polyA sequence. p-EV is identical to the p-mCAT construct, except lacking the EGFP and mCAT sequences.
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