Pcdna3 n flag asc1

NLRP3 inflammasomes were reconstituted in 293T cells to explore the ROP7 functioning.
To analyze IL-1β secretion, the 293T cells were seeded in 24-well plates and co-transfected with 15-ng pcDNA3-N-Flag-NLRP3 (#75127, Addgene, Cambridge, MA, USA), 5-ng pcDNA3-N-Flag-ASC1 (#75134, Addgene, Cambridge, MA, USA), 100-ng pCMV-pro-IL-1β, and 2.5-ng pCMV-pro-caspase-1-FLAG through Lipofectamine 3000 (L3000015, Invitrogen, Carlsbad, CA, USA). For the analysis of IL-1β cleavage, the 293T cells were seeded in six-well plates and co-transfected with 150-ng pcDNA3-N-Flag-NLRP3, 50-ng pcDNA3-N-Flag-ASC1, 1000-ng pCMV-pro-IL-1β, and 25-ng pCMV-pro-caspase-1-FLAG. Different doses of pcDNA3.1-HA-ROP7 or empty vectors were transfected simultaneously. Cells and cell-free medium were collected 36 h after transfection. IL-1β secretion and cleavage were measured via ELISA and Western blot.

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.