Imagequant las 4000mini system

Preparation of whole cell lysates and Western blotting (WB) were performed as described previously^{37 (link)}. Chemiluminescent signals were detected by ImageQuant LAS4000mini system (Cytiva, Marlborough, MA, USA) or X-ray films (Fujifilm, Tokyo, Japan). Full scan images of all western blots (original blots), including replicates, were shown in Fig. S8. Most of the blots were cut prior to hybridization with antibodies, and their membrane edges failed to be visible because of high signal-to-noise ratio for hybridization with antibodies by successful pre-blocking with TBST + 5% skim milk at room temperature for 1 h.

Protein samples were subjected to SDS-PAGE (10% polyacrylamide) and electroblotted onto an Immobilon-P membrane (Millipore). We run two SDS-PAGE gels for each experiment; one was for TP53 detection and the other was for α-Tubulin. To both gels, the same amounts of the identical protein samples were loaded for normalization of TP53 expression levels using α-Tubulin as a control. Each blot was incubated overnight at 4 °C with an appropriate primary antibody as follows: anti-TP53 (1:2000, clone DO-1, sc-126, Santa Cruz Biotechnology) or anti-α-Tubulin (1:5000, clone 10G10, 017-25031, Wako). Next, each blot was then incubated with peroxidase-labeled anti-mouse IgG (1:5000, NA931V, GE Healthcare). Immunoreactive proteins were detected with Immobilon Western Chemiluminescent HRP substrate (Millipore) and the ImageQuant LAS 4000mini system (Cytiva) using Immunoblot image acquisition: Image Quant LAS 4000mini software (Cytiva). The band intensities were determined and expressed as pixel densities using ImageJ^{43 (link)} software (National Institutes of Health, Bethesda, MD), and these values were normalized against the intensity of the corresponding control (α-Tubulin). Uncropped and unprocessed scans of the blot images presented in the figures are supplied in the Source Data file.

Total protein lysates from cells were prepared using sonication with PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seongnam, South Korea). Protein levels were determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equivalent amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to membranes. The membranes were subsequently incubated with primary antibodies for 24 h at 4 °C. Primary antibodies: anti-VP3, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), STING, Phospho-STING, TBK1/NAK, Phospho-TBK1/NAK, IRF3, Phospho-IRF3 (Cell Signaling, Denver, MA, USA). Thereafter, secondary Abs, goat anti-mouse IgG F(ab′)2, polyclonal Abs (HRP conjugated) (Enzo Life Sciences, Farmingdale, NY, USA), and anti-rabbit IgG (HRP-linked antibody) (Cell Signaling, Denver, MA, USA), were added for 2 h at 20 °C. Proteins were detected using West Femto Maximum Sensitivity Substrate (Abbkine, Atlanta, GA, USA) and visualized using an ImageQuant LAS 4000 Mini System (Cytiva, Marlborough, MA, USA). Chemiluminescence intensity was analyzed using ImageJ software (NIH, Bethesda, MD, USA).