Anti tlr3

Cell lysates were prepared in cell lysis buffer (Cell Signaling, Danvers, MA, USA). Protein concentration was determined by the micro bicinchoninic acid method (Pierce, Rockford, IL, USA).
Equal amounts of proteins (25–40 μg) were subjected to SDS-PAGE and transferred onto nitrocellulose membrane. Then membranes were saturated with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 and incubated overnight with primary antibody at 4°C and subsequently with horseradish peroxidase-conjugated secondary antibody for 1 hr at room temperature. Membranes were washed with Tris-buffered saline with 0.1% Tween-20 after each antibody incubation and developed by using the chemiluminescence system (ECL Advance; Amersham Bioscience, Piscataway, NJ, USA). Source of primary antibodies: anti-phospho-IRF-3, anti-TLR3 and anti-Cleaved Caspase 3 from Cell Signaling (Beverly, MA, USA), anti-NOXA from Enzo Life Science (Lausen, Switzerland) and β-actin antibodies from Sigma-Aldrich. Horseradish peroxidase-conjugated goat antimouse or anti-rabbit Secondary antibodies were from Bio-Rad (Hercules, CA, USA).

Cultured epithelial cells after stimulation were rinsed with DPBS and proteins were extracted using radio-immunoprecipitation assay (RIPA) buffer (GenDEPOT). Denatured proteins (25 μg) were fractionated on SDS-PAGE and the gels were transferred onto a polyvinylidene difluoride membrane (Bio-Rad, MA, USA). Thereafter the membranes were rinsed and probed with the primary antibodies in the refrigerator overnight at 4 °C; the primary antibodies employed for Western blots were anti-β actin (1:5000) which was obtained from Santa Cruz, USA, anti-viperin, anti-OAS, anti-Mx, anti-TLR3, anti-RIG1, anti-MDA5, anti-SOD1, anti-SOD2, anti-IRF3, and anti-phospho-IRF3 which were obtained from Cell Signaling Technology and used at a dilution of 1:1000.

Immunoblotting was carried out as described previously13 (link). The primary antibodies used were anti-RIG-I (IMGENEX), mAb anti-phospho IRF3 (Ser396), anti-TLR3 (Cell Signaling Technology, Beverly, MA), anti-IRF3 and anti-actin (Sigma).