Anti sting

Cells were lysed in SDS sample buffer, denatured at 95°C for 10 min, sonicated for 10 min, and lysates were separated by 10% SDS-PAGE. After transfer to nitrocellulose membranes, membranes were blocked in 5% milk (TBS, 0.1% Tween) before incubation with anti-cGAS (1:1,000; Sigma or 1:1,000; Cell Signaling), anti-STING (1:5,000; R&D Systems or 1:1,000; Cell Signaling), anti-IFI16 (1:2,000; Santa Cruz), anti-P-IRF3 (1:2,000; Cell Signaling), or anti-IRF3 (1:2,000; Cell Signaling) antibodies over night or anti-β-Actin-Peroxidase (1:50,000; Sigma-Aldrich) antibody for 1 h. After 2 h of incubation with secondary goat anti-rabbit-HRP, and goat anti-mouse-HRP (KPL), or goat anti-mouse IgG1-HRP (Southern Biotech) membranes were developed with Amersham ECL Western Blotting Detection Reagent (GE Healthcare). For detection of P-IRF3 SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) was mixed 1:10 with Amersham ECL Western Blotting Detection Reagent.

HFF cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing a protease and phosphatase inhibitor cocktail (Pierce Biotechnology, Waltham, MA). Equal amounts of protein (20 to 40 μg, depending on the target protein) were resolved in 8 to 10% (wt/vol) SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (PALL, Cortland, NY). The membranes were blocked with TBST (150 mM NaCl, 10 mM Tris-HCl, 0.1% [vol/vol] Tween 20, pH 8.0) containing 5% (wt/vol) skim milk and analyzed with the following primary antibodies: anti-YAP (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, sc-101199), anti-STING (1:500, R&D systems, Minneapolis, MN, MAB7169), anti-IE1 (1:1000, Millipore, Billerica, MA, MAB8131), anti-pp52 (1:1000, Virusys, Sykesville, MD, ICP36), anti-pp28 (1:1000, Santa Cruz Biotechnology, sc-69749), and anti-β-actin (1,10,000, Santa Cruz Biotechnology, sc-47778). All blots were incubated overnight with primary antibody at 4°C with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies for 2 h at room temperature. The enhanced chemiluminescence system (Atto, Tokyo, Japan) and X-Omat film (Kodak, Rochester, NY) were used to visualize the protein bands.