Pgl3 nf κb

RK-13 cells were plated in 24-well plates for 12 h prior to transfection. The luciferase reporter plasmids pGL3-NF-κB, pGL3-IFN-β, and pGL3-ISRE were purchased from Agilent (Santa Clara, CA, USA). The pRL-TK plasmid (Promega, Madison, WI, USA) acted as an internal control to normalize transfection efficiency. Cells were transfected with reporter plasmid (100 ng/well) or pRL-TK plasmid (50 ng/well) and 1 µg of pC-rNOD1, pC-rNOD1-CARD, pC-rNOD1-delCARD, or pCDNA3.1-empty or with 500 ng of si-rNOD1 or NC siRNA for 24 h using TransIL-LT1 Transfection Reagent. After 24 h of cotransfection with NF-κB, the cells were infected with 1 × 10⁷E. coli for 2 h. The medium was then removed, and the cells were cultured in DMEM containing gentamicin (100 µg/mL) for 3 h. After lysing and harvesting cells, luciferase activities were detected with a dual-luciferase reporter assay system (Beyotime, Wuhan, China).

The RK-13 cells were cultured in 24-well plates and grown in standard conditions for 12 h prior to transfection. The luciferase reporter plasmids (pGL3-NF-κB and pGL3-IFN-β) were purchased from Agilent (Santa Clara, CA, USA). The pRL-TK plasmid (Promega, Madison, WI, USA) acted as an internal control to normalize transfection efficiency. 1 μg of pC-rNOD2 or pCDNA3.1 (+) vector and 500 ng of si-rNOD2 or NC siRNA with 100 ng of reporter plasmid and 50 ng of pRL-TK plasmid were transfected into cells using TransIL-LT1 Transfection Reagent. After 24 h, the cells were lysed and harvested, and luciferase activities were examined by a dual-luciferase reporter assay system (Beyotime, Wuhan, China).