P irf3 clone 4d4g

Cells were lysed on ice in RIPA buffer (ThermoFisher) with protease inhibitors and protein concertation was determined by BCA assay (ThermoFisher). 30 µg of total protein was mixed with 4X sample buffer containing 10% beta-mercaptoethanol and incubated at 65°C for 15 min. Samples were loaded onto a 4–15% TGX gel (Biorad, Hercules, CA) and then transferred to a PDVF membrane. Blocking was carried out in TBST with 1% fish gelatin and 1.5% polyvinylpyrrolidone (PVP). Primary antibodies used included: IRF3 (clone: D83B9, 1:100 dilution, Cell Signaling), p-IRF3 (clone: 4D4G, 1:1000 dilution, Cell Signaling), and GAPDH (1:6000 dilution, Novus Biologicals). Primary antibodies were incubated with the membrane overnight at 4°C with rocking. Membranes were then incubated for 1 hr in secondary antibodies at room temperature before exposure to Clarity Western ECL substrate (Biorad) and imaging on the Azure Imaging System (Azure Biosystems, Dublin, CA). Membranes are then stripped, and reprobed with anti-GAPDH. Signal intensity was quantified by densitometry using AzureSpot Analysis Software (Azure Biosystems), with ratios of the band of interest/housekeeping gene used to normalize variability in protein loading.