Double luciferase reporter gene detection kit

The expression plasmid pCMV-Tag 2B was used to amplify the target fragment with primers containing restriction sites. The ORF of LmTLR14d was digested with BamHI and EcoRI (Supplementary Table 1). The sequence of expression plasmid was identified by Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing. The reporter gene of LmNF-κB promoter (accession number MN368861) was linked to reporter vector pGL3-Enhancer plasmid. LmMyD88 and LmTRIF were cloned into pCMV-Tag 2B plasmid, respectively. HEK293T cells were seeded in 24 well plates at 8×10⁴ cells per well for 24 h to transfect. The pCMV-LmTLR14d, pCMV-LmTLR14d LRR, pCMV-LmTLR14d TIR, pCMV-MyD88, pCMV-LmTRIF and NF-κB reporter gene of L. morii were co-transfected into cells in different groups with Fugene HD transfection reagent, and phRL-TK plasmid was used as the internal reference. After 36 h of transfection, the activities of firefly luciferase and Renilla luciferase were detected using a double luciferase reporter gene detection kit (Promega), and the biology was repeated three times.