Anti p irf3

LLC-PK1 or HEK-293T cells were cultured in 60-mm dishes, transfected with the indicated plasmids for 24 h, and then infected or mock-infected with SEV (50 hemagglutinating activity units/dish) for 8 h. The cells were harvested by adding lysis buffer (4% SDS, 3% dithiothreitol [DTT], 0.065 mM Tris-HCl [pH 6.8], 30% glycerin) supplemented with a protease inhibitor cocktail, phenylmethylsulfonyl fluoride (PMSF), and a phosphatase inhibitor cocktail (Sigma). The samples were subjected to separation by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) to determine protein expression. The endogenous expression of p-IRF3, p65, p-p65, and IRF3 proteins were analyzed with the indicated antibodies, anti-p-IRF3, anti-p65 (ABclonal), anti-p-p65, and anti-IRF3 (Cell Signaling Technology), respectively. The overexpression of a plasmid with a HA tag was evaluated with an anti-HA antibody (MBL), and the expression levels of β-actin were detected with an anti-β-actin monoclonal antibody (MBL). The detections of similar amounts among groups were taken to indicate equal protein sample loading.