The pReceiver-M56-DDX3X-mCherry expression clone was purchased from GeneCopoeia. DDX3X-ΔNterm (residues 156–662), DDX3X-ΔCterm (residues 1–573) and DDX3X-helicase (residues 156–573) constructs were generated in the same vector background. NLRP3 expression constructs pCDNA3-N-Flag-NLRP3, pCDNA3-N-Flag-NLRP3 1–90, pCDNA3-N-Flag-NLRP3 91–710 and pCDNA3-N-Flag-NLRP3 711–1034 were purchased from AddGene. To interrogate domain specificity of DDX3X and NLRP3, HEK293T cells were transiently transfected with 4 μg of the full-length DDX3X or NLRP3 construct in combination with 4 μg of one of the domain constructs of NLRP3 or DDX3X using Xfect (Takara) or Lipofectamine-2000 (Invitrogen) in Opti-MEM (Gibco). Opti-MEM was replaced with DMEM with 10% FBS 8 h after transfection, and 24–30 h after transfection, cells were lysed with NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris and protease-phosphatase inhibitor cocktail). Whole-cell lysates were centrifuged to separate the insoluble fraction, and the soluble fraction was further subjected to immunoprecipitation experiments. Antibodies for mCherry (Abcam), Flag-M2 (Sigma) and NLRP3 (AdipoGen/CST) were used to detect DDX3X and NLRP3 on immunoblots.
Pcdna3 n flag nlrp3
Manufactured by Addgene
Sourced in United States
The pcDNA3-N-Flag-NLRP3 is a plasmid vector that expresses the NLRP3 (NOD-like receptor family, pyrin domain-containing 3) protein with a Flag tag at the N-terminus. The NLRP3 protein is a key component of the inflammasome, a multiprotein complex involved in the innate immune response.
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Lab products found in correlation
Pcdna3 n flag asc1, by Addgene (2 mentions)
Xfect, by Takara Bio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Flag m2, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
4 protocols using pcdna3 n flag nlrp3
1
Interactome Analysis of DDX3X and NLRP3
2
ROP7 Modulates NLRP3 Inflammasome Activation
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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 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.
3
NLRP3-Inflammasome Expression Plasmids
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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.
4
Transfection of HEK293 cells with plasmids
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Human embryonic kidney cells (HEK293) were grown in 6 wells plates in 25 mM D-glucose DMEM supplemented with 10% fetal calf serum (Sigma®). The OGT and OGA plasmids were previously described (10 (link)), the TxNIP plasmid was purchased from Genecust® and pcDNA3-N-Flag-NLRP3 was a gift from Bruce Beutler (Addgene plasmid # 75127). Transfections of HEK293 cell were performed using Lipofectamine 2000 and OptiMEM, and 1 μg of plasmid/well.
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