Irf 3 clone d83b9

BMDM were washed twice with Hanks Buffered Saline Solution (HBSS) and lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing PhosStop and complete Protease Inhibitor tablets (Roche). Protein samples were mixed 1:1 with Laemmli buffer (Bio-Rad) and separated by gel electrophoresis on MINI-PROTEAN precast 4–20% tris-glycine gels (Bio-Rad). Protein was transferred to nitrocellulose membranes (Perkin-Elmer). Membranes were blocked with 5% fraction V bovine serum albumin (BSA) (RPI Corp.) for one hour and incubated with primary antibodies from Cell Signaling Technology (1:1000 dilution; phospho-IKK-alpha/beta [Clone 16A6], Cat:2697S; IKK-beta [clone 2C8], Cat: 2370S; Phospho-IRF-3 [clone S396], Cat: 4947S; IRF-3 [clone D83B9], Cat: 4302S) in 5% BSA at 4°C overnight. Membranes were washed 3 times for 5 minutes in tris-buffered saline with 0.1% Tween-20 (TBST). Protein bands were detected with HRP-conjugated secondary antibodies (1:2000 dilution; anti-Rabbit IgG, HRP-linked, Cat: 7074S) in 5% BSA, washed 3 times for 5 minutes in TBST, and incubated for 60 seconds with electrochemiluminescence (ECL) reagent. Images were cropped using ImageJ 1.52a software from the NIH (http://imagej.nih.gov/ij/).

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.