Pgl4.32 luc2p nf κb re hygro plasmid

293A cells were cultured overnight in a 10-cm culture dish and then co-transfected with 7.5 μg of pUNO1-hTLR5 plasmid (InvivoGen) and 5.5 μg of pGL4.32 [luc2p/NF-κB-RE/Hygro] plasmid (Promega) using TurboFect transfection reagent (Fermentas). The next day, the transfected cells were seeded at a density of 5 × 10⁴ cells/well in a 96-well plate, followed by the addition of a 10-fold serial dilution of the recombinant FliC protein or the recombinant FliC-DIII fusion proteins, which were first diluted to 1 μg/ml in DMEM. After incubating for 5 h, the cells were disrupted using Glo lysis buffer (Promega) and treated with Neolite luciferase substrate (PerkinElmer). After 5 min, a VICTOR3 Multi-labeled Microplate Reader (PerkinElmer) was used to read the 96-well plate for the measurement of the relative luminescence units (RLUs).

AGS cells stably expressing a tdTomato-TIFA-Flag construct (tdTomato construct from Takara, Kyoto, Japan) were created using the pMW1064 (td-Tomato/TIFA-Flag/Kan) plasmid. The tdTomato-TIFA-Flag gene was shuttled in a lentiviral expression plasmid (pLenti ×1 Zeo Dest, Plasmid 17299; Addgene, Watertown, MA, USA). Cells were lentivirally transduced and selected with zeocin, and a cell line was created from single clones. AGS cells stably expressing an NF-κB-luc2P construct were created using the pGL4.32 (luc2P/NF-κB-RE/Hygro) plasmid (Promega, Madison, WI, USA). The NF-κB response element and the luc2P reporter gene was shuttled in a pLenti plasmid (pLenti phosphoglycerate kinase Neo Dest, Plasmid 19067; Addgene). Cells were lentivirally transduced and selected with neomycin, and a cell culture was created from single clones. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) control and ALPK1 or TIFA knockout cells were created as previously described by Zimmermann et al. (9 (link)) from the AGS parent cell line [1739; American Type Culture Collection (ATCC), Manassas, VA, USA]. All cells were cultivated at 37°C and 5% CO₂ in Roswell Park Memorial Institute medium (Thermo Fisher Scientific, Waltham, MA) with 10% heat-inactivated fetal calf serum (9 (link)).