Rabbit monoclonal anti sting

Western blotting was performed as previously described (14 (link)) with minor modifications. Cellular proteins were isolated using 1 × RIPA buffer containing a protease inhibitor and a phosphatase inhibitor. The proteins were resolved by electrophoresis in a 10–15% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (GE Healthcare). The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20. Rabbit monoclonal anti-STING (Cell Signaling Technology, Beverly, MA), Rabbit monoclonal anti-cGAS (Cell Signaling Technology), rabbit polyclonal anti-Rab27b (Bioss Antibodies Inc., Woburn. MA), mouse monoclonal anti-KSHV ORF65 (14 (link)), rabbit polyclonal anti-GAPDH (Cusabio, Houston, TX), rabbit polyclonal anti-calnexin (Bioss Antibodies Inc.), mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-mtTFA (Santa Cruz Biotechnology), rabbit polyclonal anti-MX1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT44L (Bioss Antibodies Inc.) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies. HRP-conjugated anti-rabbit or anti-mouse antibodies (Bethyl Laboratories Inc., Montgomery, TX) were used as secondary antibodies. The results were visualized using an ECL detection reagent (Bio-Rad, Hercules, CA).

Samples were resolved by SDS–PAGE and transferred onto a 0.45-μm nitrocellulose membrane. Rabbit monoclonal anti-GAPDH (2118, 1:5,000; Cell Signaling), mouse monoclonal anti-IRF3 (50772, 1:100; Abcam), rabbit monoclonal anti-TBK1 (3504, 1:1,000; Cell Signaling), and rabbit monoclonal anti-STING (13647, 1:1,000; Cell Signaling) blots were blocked in 5% nonfat powdered milk dissolved in Tris-buffered saline containing 0.1% Tween (TBST). Rabbit monoclonal anti–phospho-IRF3 (Ser396 4947, 1:1,000; Cell Signaling), rabbit monoclonal anti–phospho-STING (Ser366 19781, 1:1,000; Cell Signaling), rabbit monoclonal anti–phospho-TBK1 (Ser172 5483, 1:1,000; Cell Signaling), and rabbit monoclonal anti-phospho-H3 (Ser10 3377, 1:10,000; Cell Signaling) blots were blocked in 100% Odyssey Blocking Buffer (927–40000; LI-COR). Goat antirabbit DyLight 680 (3568; Pierce), goat antimouse DyLight 680 (35518; Pierce), goat antirabbit DyLight 800 (535571; Pierce), and goat antimouse DyLight 800 (35521; Pierce) were used as secondary antibodies at 1:10,000 in either 50% Odyssey Blocking Buffer/TBST or 5% milk/TBST. Blots were imaged with the Licor Odyssey Infrared Imaging System. Band intensities were quantified by densitometry using ImageJ v1.52a (104 (link)).