Amersham imager 680

Cell lysis was performed in RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitor cocktail (Roche). Samples were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare). Antibodies were obtained from Cell Signaling Technology: STING (#13647), anti-rabbit HRP conjugated (#7074), and Sigma-Aldrich: β-actin (#A3854). The blots were visualized with Clarity and Clarity Max substrates (Bio-Rad) using Amersham Imager 680 (GE Healthcare).

The frozen liver tissue samples were homogenized in a lysis buffer containing 0.1 mM sodium vanadate, a protease inhibitor cocktail tablet (Roche, Mannheim, Germany), pefabloc SC (Roche, Mannheim, Germany), sodium fluoride, and sodium pyrophosphate. The whole liver lysates were measured using a DC Protein Assay Kit (Bio Rad, Hercules, CA, USA). Equal amounts of protein (50 μg) were loaded and separated by 10% of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the protein samples were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, IPVH00010, Billerica, MA, USA). The membranes were blocked in 5% skim milk for 1 h and incubated with the primary antibodies overnight at 4 °C. After washing with Tris buffered saline containing 0.1% Tween-20, membranes were incubated with a horseradish-peroxidase-conjugated goat-anti-rabbit (Calbiochem, 401393, San Diego, CA, USA) or goat-anti-mouse (Calbiochem, 401253, San Diego, CA, USA) antibody for 1 h at room temperature. The membranes were visualized using the ProNA™ ECL Ottimo (Translab, Seoul, Korea), and the images were acquired using the Amersham™ Imager 680 (GE Healthcare, Bjorkgatan, Sweden). β-actin was used as a loading control.

A549 cells were treated with 1 µmol/L MLN4924 for either 12 h or 24 h. As a control, an equal volume of DMSO was added. Cells from each group were collected, lysed with sodium dodecyl sulfate buffer, and heated at 100 °C for 10 min. Proteins were separated on 10% Bis–Tris polyacrylamide gels by electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, USA). Protein bands were visualized using an enhanced chemiluminescence kit and images were captured using an Amersham Imager 680 (GE Healthcare, IL, USA). Anti-Cullin1 antibody (Abcam, Cambridge, United Kingdom, 75817), anti-phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (p-IκBα) (Cell Signaling Technology, MA, USA, 2859), anti-IκBα (Cell Signaling Technology, MA, USA, 9242) and anti-β-actin antibody (HuaAn, M1210-2) were purchased.

Cell lysis and immunoblot analysis were performed as described previously.⁵⁶ (link)^,57 (link) Briefly, the samples were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). Western blotting was performed and the bands were visualized with the Amersham ECL Select Western blotting detection reagent and an Amersham Imager 680 (GE Healthcare). The band intensities were quantified by Amersham Imager 680 Analysis software (GE Healthcare).

EMSA experiments were performed as described previously with minor modifications (25 (link)). The 5′-fluorescein-labeled 24 bp dsDNA (5′-GGATGCATGAGTACGAGGACCATC-3′ (the specific probe) and 5′-GGATGCATGAGATCGAGGACCATC-3′ (the nonspecific probe)) were used as probes (Supplementary Figure S1BC). Then, 0.1 μM of the DNA probe and the R.PabI (Y68F and its mutants) dimer were mixed in 10 mM MES pH 6.0 and 300 mM NaCl in the presence of a 25-fold excess amount of an unlabelled competitor dsDNA (5′-GGATGCATGAGATCGAGGACCATC-3′). In addition, 0.1 μM of the DNA probe and the R.PabI (K154A and its mutants) dimer were mixed in the same buffer in the presence of a 50-fold excess amount of the unlabeled competitor dsDNA. The samples were separated using a 10% polyacrylamide gel in 0.5 × TBE at 4°C. The fluorescence was measured using an Amersham Imager 680 (GE Healthcare) and was quantified with Amersham Imager 680 Analysis Software (GE Healthcare).