Dual light reporter gene assay system

TLR10 complementary DNA (cDNA) was cloned into a pCMV6 vector (OriGene, Rockville, MD, USA). The I473T variant was generated by site-directed mutagenesis using the QuikChange mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) with the following primers: 5′-GGCCTTACGAGAACTAAATACTGCATTTAATTTTCTAACTGATC-3′ and 5′-GATCAGTTAGAAAATTAAATGCAGTATTTAGTTCTCGTAAGGCC-3′. The modified insert was sequenced to verify the mutation. K562 and U937 cells were cotransfected with 2 μg of wild-type or mutant TLR10 constructs, 1 μg of reporter plasmid pBVIx-Luc, containing six tandem repeats of the NFkB recognition sites within the promoter region linked to the luciferase gene [34 (link)] and 0.2 μg of respiratory syncytial virus-β-galactosidase plasmid (pRSV-β-gal) by using Lipofectamine 2000 reagent (Sigma-Aldrich, St. Louis, MO, USA). After 24 h of transfection, cell viability was greater than 80 %. Then, K562 and U937 cells were incubated with 10 ng/ml and 20 ng/ml tumor necrosis factor-α (TNFα) in the presence or absence of 200 μg/ml infliximab, and, after 24 h, cell extracts were prepared and analyzed to measure relative luciferase activity by using a dual-light reporter gene assay system (Applied Biosystems). Results were normalized for transfection efficiency with values obtained with pRSV-β-gal.