Knockdown of NF-κB p65 with siRNA.

Silencing RNAs directed toward porcine NF-κB p65 mRNA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_001114281.1","term_id":"166796054"}}NM_001114281.1) were customized using a design small interfering RNA (siRNA) tool from Integrated DNA Technologies (IDT). Scrambled negative-control siRNAs (catalog number 51-01-19-09; IDT) and NF-κB p65 siRNAs (5′-GCAUCAUGAAGAAGAGUC-3′; 5′-UUGAAAGGACUCUUCUUC) were also synthesized by IDT. The annealing of the two RNA strands was performed by incubating the RNAs at 95°C for 2 min, and then the RNAs were cooled to room temperature to reach the optimal annealing temperature. The transfection of preconfluent ST cells (confluence, 70 to 80%) was done by mixing 2.5 μl of siRNA suspension (10 μM, diluted in nuclease-free duplex buffer [IDT]) and 7.5 μl of the Lipofectamine RNAiMAX reagent (catalog number 13778030; Thermo Fisher Scientific) in Opti-MEM medium per well of a 6-well plate. The siRNA and lipofectamine volumes were reduced one-fourth to adapt the protocol to a 24-well-plate system. The transfection procedure was carried out according to the manufacturer’s indications. Cells were incubated with Lipofectamine reagent-RNA complexes for 48 h, and then they were subjected to virus infections. The effectiveness of NF-κB p65 knockdown was assessed by Western blotting with a specific anti-NF-κB p65 antibody (catalog number 6956; CST).