Pnf κb luc plasmid

The NF-kB promoter activity was evaluated using the luciferase assay kit (Promega, Madison, WI, USA) as described previously [57 (link)]. Cells were transfected with the pNF-κB-Luc plasmid (Clontech). Following treatment, luciferase assay solution was added, and the luminescence was determined using a luminometer. The promoter luciferase activities were standardized to β -galactosidase.

The pLKO.1-mCherry-Puro plasmid was kindly provided by Dr Renzhi Han of the Ohio State University. The target short interfering RNA sequences was identified using BLOCK-iT RNAi Designer online software (Life Technologies). At least two short interfering RNA sequences were tested for efficient knockdown of target TAK1 mRNA. The shRNA oligonucleotides were synthesized to contain the sense strand of target sequences for mouse TAK1 (that is, 5′-GGTGCTGAACCATTGCCTTAC-3′), short spacer (CTCGAG) and the reverse complement sequences followed by five thymidines as an RNA polymerase III transcriptional stop signal. Oligonucleotides were annealed and cloned into pLKO.1-mCherry-Puro with AgeI/EcoRI sites. The insertion of shRNA and complementary (cDNA) sequence in the plasmids was confirmed by DNA sequencing. Constitutive active mutant of IKKβ (that is, IKK-2 S177E S181E) was a gift from Professor Anjana Rao (Addgene plasmid # 11105). pCDNA3 Flag MKK7B2Jnk1a1 was a gift from Roger Davis (Addgene plasmid # 19726). pNF-κB-Luc plasmid was purchased from Clontech. pRL-TK plasmid was from Promega (Madison, WI).

Estrogen Response Element activation. SKBR3 and MCF7 cells were cultured in 24-well plates and transfected with 0.2 μg/well of either the ERE-tk-Luc plasmid or its control tk-Luc plasmid. The first one carried estrogen response elements in front of the 5′ end of a minimal thymidine kinase promoter and the firefly luciferase gene, while the second one lacked the EREs.
NFκB activation assay. SKBR3 and MCF7 cells were cultured in 24-well plates, were transfected with 0.2 μg/well of the pNFκB-Luc plasmid (Clontech, Mountain View, CA, USA), carrying NFκB response elements in front of the 5′ end of the firefly luciferase gene.
In all cases, cells were also transfected cells with 0.2 μg/well of a Renilla luciferase vector (pRL-CMV, Promega, Fitchburg, WI, USA). Lipofectamine 2000 (Invitrogen, 1 μL/well) in Optimem medium was used in all transfections. Cells in both cases were incubated for 48 h before treatment. Luciferase activity was assayed with a Dual-Luciferase Reporter 1000 Assay System (Promega, Fitchburg, WI, USA), in a Berthold FB12 Luminometer (Bad Wildbad Germany).