Anti irf3 rabbit polyclonal antibody

For Immunoblot, whole cell lysate was obtained with low-salt lysis buffer after virus infection for indicated time points. Protein samples were mixed with the 5X loading buffer (Cell Signaling Technology) and resolved by SDS-PAGE. After electrophoresis, protein was transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories) and then incubated with appropriate antibody. LumiGlo Chemiluminescent Substrate System (KPL) was used to detect specific band of certain protein. Antibodies used in immunoblot were listed as follows. Anti-IRF3 rabbit polyclonal antibody, goat anti–mouse IgG-HRP and goat anti–rabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology Inc. Anti-phospho-IRF3 (Ser396) rabbit monoclonal antibody, anti-RIG-I rabbit monoclonal antibody, anti-p38 MAPK rabbit polyclonal antibody, anti-phospho-p38 MAPK (Thr180/Tyr182) rabbit polyclonal antibody, anti-SAPK/JNK rabbit polyclonal antibody, anti-phospho-SAPK/JNK (Thr183/Tyr185) rabbit polyclonal antibody, anti-TBK1/NAK (D1B4) rabbit monoclonal antibody, anti-phospho-TBK1/NAK (Ser172) rabbit monoclonal antibody, IκBα muse monoclonal antibody were purchased from Cell Signaling Technology. Anti-IRF1 mouse polyclonal antibody was bought from abcam^®.

Cell lysates were prepared by scraping cells into PBS followed by centrifugation at 200 × g for 5 min and resuspension in native PAGE lysis buffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl and 1 % NP-40) containing 1X protease and phosphatase inhibitor cocktail. Non-denaturing 7.5 % acrylamide gels were pre-run at 40 mA constant current for 30 min in cathode buffer (25 mM Tris-Cl, 192 mM glycine and 0.2 % DOC, pH 8.4) and anode buffer (25 mM Tris-Cl,192 mM glycine, pH 8.4) at room temperature and equal quantities of proteins were separated at 25 mA constant current for 50 min at 4 °C [40 (link)]. Proteins were transferred to PVDF membrane at a constant current of 350 mA for one hour at 4 °C. IRF-3 monomers and dimers were detected using an anti-IRF-3 rabbit polyclonal antibody (Santa Cruz Biotechnology: sc-9082) followed by an HRP conjugated secondary antibody and chemiluminescence.