Anti irf3 clone fl 425

Guanidine whole-cell lysates were precipitated using a ProteoExtract protein precipitation kit (Calbiochem). Proteins were re-dissolved in 2% SDS/Tris 200 mM pH8.5 with sonication. Protein concentration was measured by BCA (Pierce) then lysates were reduced with 100 mM DTT in loading buffer for 20 min at 65°C. Equal protein amounts were separated by SDS/PAGE using 4%–12% Bis-Tris polyacrylamide gels (Life Technologies), then transferred to PVDF membranes using the iBlot transfer system (Life Technologies). The following primary antibodies were used: anti-IFIT1 (clone 3G8, Lifespan), anti-IFIT3 (cat no. GTX112442, GeneTex), anti-MX1 (clone 2G12, GeneTex), anti-Calnexin (cat no. C142768, Lifespan), anti-MHC class I (clone HC-10, Abcam), anti-CD99 (cat no. ab108297, Abcam), anti-CLEC1A (cat no. AF1704, R and D systems), anti-FAT1 (cat no. HPA023882, Sigma), anti-PCDHGC3 (cat no. sc-134416, Santa Cruz), anti-IRF3 (clone FL-425, Santa Cruz), anti-NF-kB p50/105 (cat no. sc-7178, Santa Cruz), anti-p65/RELA (cat no. sc-109, Santa Cruz), anti-Pan-Akt (clone C67E7, Cell Signaling), anti-actin (cat no. A2066, Sigma). HRP-conjugated secondary antibodies were anti-rabbit (Cell Signaling), anti-mouse (Santa Cruz) or anti-goat (Santa Cruz). Reactive bands were detected by SuperSignal West Pico substrate (Thermo).

For confirmation of targeted knockdown experiments as well as transient transfection/reconstitution experiments in both murine and human cell lines, the following primary antibodies were used: anti-hMAVS (sc-166583; Santa Cruz Biotechnology), anti-mMAVS (#4983; Cell Signaling Technology), anti-hRIG-I (70R-16795; Fitzgerald Industries International), anti-mRIG-I (#3743; Cell Signaling Technology), anti-MDA5 (#5321; Cell Signaling Technology), anti-LGP2 (70R-16832; Fitzgerald Industries International), anti-TBK1 (sc-9910; Santa Cruz Biotechnology), anti-phospho-TBK1 (S172 clone D52C2; 5483S; Cell Signaling Technology), anti-IRF-3 (clone FL-425; sc-9082; Santa Cruz Biotechnology), anti-FLAG (M2 clone; Sigma), anti-HA (Y-11 clone; sc-805; Santa Cruz Biotechnology), anti-α-Tubulin (sc-12462R; Santa Cruz Biotechnology), and anti-Actin-HRP (sc-47778; Santa Cruz Biotechnology). Secondary antibodies conjugated to HRP (Santa Cruz Biotechnology) were used at a 1:10,000 dilution.