COS-7 cells (product no. CRL1651, ATCC) in 96-well plates were cultured in Dulbecco’s modified medium supplemented with 10% FBS and penicillin/streptomycin, and transiently transfected with a reporter plasmid [pCRE-luc, pNFAT-luc, or pNFκB-luc (Clontech)], an internal control plasmid (pRL-EF1α) [47 (link)], and an effector plasmid (pcDNA-GP33s and -GP33) or positive control plasmids, by adding 200-fold diluted lipofectamine 2000 (Invitrogen) and cultured for 6 h. Total amount of DNA for each condition was adjusted by adding the empty vector. The cells were then cultured in a serum-free medium for 24 h and rinsed with PBS. The cells were treated with a Dual-luciferase reporter assay kit (Promega), and the luciferase activities of the cells were measured by GloMax (Promega). pF4A-CMV-GαqQ209L and a plasmid expressing FLAG-MyD88, kindly provided by H. Ueda (Gifu Univ.) and A. Takaoka (Hokkaido Univ.), respectively, were used as positive control plasmids for the NFAT- and NFκB-pathways, respectively.
Pnf κb luc
Manufactured by Takara Bio
Sourced in United States, Japan, Canada
The PNF-κB-Luc is a reporter construct that contains the firefly luciferase gene under the control of a promoter element responsive to the NF-κB transcription factor. This construct can be used to monitor NF-κB activation in cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase assay system, by Promega (7 mentions)
Dual luciferase reporter assay system, by Promega (6 mentions)
Prl tk, by Promega (5 mentions)
Pap1 luc, by Takara Bio (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Phrg tk, by Promega (3 mentions)
Prsv βgal, by Addgene (3 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Prl tk, by Takara Bio (2 mentions)
Ptal luc, by Takara Bio (2 mentions)
Psv β galactosidase expression plasmid, by Promega (2 mentions)
Glo lysis buffer, by Promega (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Jetpei, by Polyplus Transfection (2 mentions)
Pcre luc, by Takara Bio (1 mentions)
Glomax, by Promega (1 mentions)
Pmscv puro retroviral vector, by Takara Bio (1 mentions)
Pmirglo vector, by Promega (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Zymosan, by InvivoGen (1 mentions)
43 protocols using pnf κb luc
1
Dual-Luciferase Assay for Transcription Factor Activation
2
Studying miR-532-3p Regulation of TRAF Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human miR-532-3p gene was PCR amplified from genomic DNA and cloned into a pMSCV-puro retroviral vector (Clontech, Japan). The 3′ UTR regions of human TRAF1, TRAF2, and TRAF4 were PCR amplified from genomic DNA and cloned into pmirGLO vectors (Promega, USA). The control plasmids and pNFκB-luc (Clontech, Japan) were applied to quantitatively examine the activity of transcription factors. Anti-miR-532-3p was purchased from RiboBio. Transfection of miRNA, siRNAs, and plasmids was performed as previously described.44 (link)
3
RANK Signaling in THP-1 Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A population of 1, 5 × 106 THP-1-derived macrophages were seeded on a 3.5 cm plate and transfected using Lipofectamine® 2000 (2 μl/ml) (Invitrogen) diluted in Opti-MEM (Gibco) with 5 μg of the luciferase reporter plasmid regulated by NF-κB, pNFκB-Luc (#631745, Clontech) or a plasmid expressing human RANK point mutated, pcDN3A-human-RANK∆, in a final volume of 1 ml per plate. The cells were transfected for 5 h, and the medium was replaced with 1 ml of supplemented RPMI. After 24 h, the cells were stimulated with recombinant RANKL, LPS, or costimulus LPS+RANKL for 24 h. The pcDN3A-human-RANK∆ plasmid was constructed by using a pcDNA3 template and insertion of the human RANK sequence (NCBI GenBank AF018253.1) point mutated at TRAF6 binding sites, 344-349 PTEDEY, 377-382 PLEVGE, and 453-458 PGEDHE by the substitution of glutamic acid (E) for alanine (A) in residues 346, 379, and 455. Luciferase activity was quantified accordingly to the manufacturer's instructions (Promega). Briefly, the cells were collected and lysed, and an equivalent protein concentration was incubated in the presence of luciferin substrate. The luminescence was measured using a 96-well plate, and data were shown as CPS—counts per second.
4
Toll-like Receptor Signaling Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The NF-κB luciferase promoter construct pNF-κB-Luc and the transfection control reporter vector pRL-TK, were purchased from Clontech Laboratories, Inc., (Mountain View, CA), and Promega (Madison, WI), respectively. Wild-type pCMV-TLR1 vector was a gift from Dr Koichi Kuwano (Kurume University School of Medicine, Kurume, Japan). pCMV-MyD88DN expression construct was a gift from Dr. Jason A. Boch (Department of Medicine, Harvard Medical School, Boston, MA). Dominant-negative pZero-hTLR2 expression plasmid, puromycin, blasticidin, zymosan, FSL-1, and Pam3Csk4 were purchased from Invivogen (San Diego, California). IgG2a Isotype control monoclonal antibody was purchased from eBioscience (San Diego, California) and anti-TLR2 (clone TL2.1) monoclonal antibody was purchased from Imgenex (San Diego, California).
5
Promoter Activity Analysis of NF-κB and IL-6
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the analysis of the promoter activity of the NFκB-responsive promoter reporter luciferase construct, the cells were transfected with pNFκB-Luc (Clontech, Palo Alto, CA) using a LipofectAMINE Reagent (Invitrogen, Carlsbad, CA), and luciferase activity was determined using a Luciferase Assay System Kit (Promega, Madison, WI).
For the analysis of il6 promoter activity, the murine il6 promoter (distal fragment, −1029 to +31; proximal fragment, −649 to +31) was amplified from mouse genomic DNA (Promega) using an LA Taq polymerase (Takara bio) and was subcloned into pCR-XL-TOPO vector (Invitrogen). The subcloned fragments were digested at KpnI/XhoI sites and cloned into pGL3 vector (Promega) at the corresponding sites. The cells were transiently transfected by using a LipofectAMINE Reagent with distal or proximal constructs containing the luciferase reporter gene, and luciferase activity was determined with a Dual Luciferase Assay System Kit (Promega). Activity was normalized relative to an internal cotransfected constitutive control (Renilla luciferase expression vector, pRL-TK vector, Promega), as described [5 (link), 10 (link), 12 (link)].
For the analysis of il6 promoter activity, the murine il6 promoter (distal fragment, −1029 to +31; proximal fragment, −649 to +31) was amplified from mouse genomic DNA (Promega) using an LA Taq polymerase (Takara bio) and was subcloned into pCR-XL-TOPO vector (Invitrogen). The subcloned fragments were digested at KpnI/XhoI sites and cloned into pGL3 vector (Promega) at the corresponding sites. The cells were transiently transfected by using a LipofectAMINE Reagent with distal or proximal constructs containing the luciferase reporter gene, and luciferase activity was determined with a Dual Luciferase Assay System Kit (Promega). Activity was normalized relative to an internal cotransfected constitutive control (Renilla luciferase expression vector, pRL-TK vector, Promega), as described [5 (link), 10 (link), 12 (link)].
6
LPS-Induced NF-κB Activation in RTH-149 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After co-transfecting RTH-149 cells with luciferase reporter plasmids pNF-κB-Luc (Clontech Laboratories, Inc.) and pRL-TK (internal control; Promega) for 48 h, cells (except control cells) were stimulated with LPS (10 μg/mL) for the indicated time periods. Then, cells were lysed with lysis buffer (Promega), and firefly and Renilla luciferase activities were measured using the Dual-Luciferase Assay System (Promega). Firefly luciferase activity was normalized to that of Renilla. In some experiments, cells were co-transfected with His-OmA20, His-OmA20-N, His-OmA20-C, His-OmA20 (C107A), or EGFP-His along with pNF-κB-Luc and pRL-TK, in order to examine the effect of OmA20 and OmA20 mutants on the activation state of NF-κB.
7
Luciferase Assay for NF-κB Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
8
NF-κB Luciferase Reporter Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The LoVo cells were seeded in 24-well plates at 1×105 cells per well. The cells were transiently co-transfected with 400 ng of pNF-κB-Luc (Clontech Laboratories, Inc., Mountain View, CA, USA) and 4 ng of pRL-SV40 (Promega, Madison, WI, USA) using Lipofectamine 2000™ (Invitrogen). The pRL-SV40 plasmid with a cDNA encoding Renilla luciferase was used as an internal control. The cell extracts were prepared in luciferase cell culture lysis buffer (Promega). The activities of firefly and Renilla luciferases were measured sequentially from a single sample with the Dual Luciferase Reporter Assay system (Promega) using a Lumat LB 9507 luminometer (Bethold Technologies, Bad Wildbad, Germany).
9
Tip110 and USP15 Plasmids and Knockdown
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tip110.His, Tip110ΔN.His, Tip110ΔC.His, GFP-Tip110ΔNLS, Tip110ΔNLS.His, and GFP-Tip110 plasmids were described elsewhere (28 (link), 74 (link)). Tip110Δ274-963.His, Tip110Δ387-963.His, Tip110Δ557-963.His, and Tip110Δ786-963.His plasmids were constructed in the backbone of pcDNA3 (Invitrogen) using standard PCR techniques with Tip110.His as a template and using EcoRI and XhoI cloning sites. USP15.Myc (1–952 aa), USP15ΔC.Myc (1–384 aa) and USP15ΔN.Myc (385-952 aa) were described elsewhere (25 (link)). NF-κB p65 was a kindly provided by Dr. Michael Klemsz of Indiana University School of Medicine. pNF-κB-luc and pAP-1-Luc were purchased from Clontech Laboratories Inc., CA. UB.HA plasmid was kindly provided by Dr. Mark Hannink of the University of Missouri. IκBα.HA plasmid was kindly provided by Dr. Michael Karin of the University of California (75 (link)). The IκBα-Nuc.Myc plasmid was constructed using IκBα.HA as a template, backbone of pCMV-Nuc-Myc (Addgene) and using SalI and NotI cloning sites. On-TARGETplus Tip110 siRNA (L-013447-01) and SiRNA control (D-001810-01) were purchased from Dharmacon.
10
NF-κB Activation Assay in RAW 264.7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW 264.7 cells (5 × 105 cells/ml) were plated in 12-well plates. The cells were transfected with 1 μg pNF-κB-Luc (Clontech, Palo Alto, CA, USA) together with 0.1 μg pRL-TK Renilla luciferase plasmid (Promega) using LipofectAMINE and PLUS reagent (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after transfection, cells were stimulated with 0, 0.1, or 10 μg/ml BAG for a further 8 h. Cells were then lysed with reporter lysis buffer (Promega), and cell lysates were assayed for firefly and Renilla luciferase activity with the Dual Luciferase Reporter Assay System (Promega) in a Victor 1420 Multilabel counter (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!