Tubulin or β actin

Cells were lysed with RIPA buffer (Sigma-Aldrich) supplemented with a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) at the specified time points. Lysate proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10–12% acrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes and blocked with 5% (w/v) skim milk in Tris-buffered saline (0.2 M Tris, 1.36 M NaCl) supplemented with 0.1% (v/v) Tween-20 (TBS-Tw) for 1 h at room temperature. This was followed by overnight incubation with primary antibodies (Cell Signaling Technology, Danvers, MA, USA) at 4 °C. As a loading control, tubulin or β-actin (Abgent, San Diego, CA, USA) antibody was used. After three washes in TBS-Tw, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies for 1 h at 25 °C. Membranes were washed with TBS-Tw and incubated in Western Lumi Pico solution (ECL solution kit; DoGen, Seoul, Korea). Signals were determined using a Fusion Solo Imaging System (Vilber Lourmat, Collégien, France). Band intensities were quantified by Fusion-Capt analysis software (Vilber Lourmat).

Immunoblot analysis and co-immunoprecipitation assays were performed as described previously (Oh and Shin, 2021 (link)). Cells were harvested and lysed with RIPA buffer (Sigma-Aldrich) containing protease and phosphatase inhibitors (Roche). Proteins were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and blocked with 5% skim milk in TBS supplemented with 0.1% Tween-20 (TBS-Tw) for 1 h at room temperature. The membranes were then incubated with primary antibodies (Cell Signaling Technology) at 4°C overnight, followed by HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies for 1 h at room temperature. Tubulin or β-actin (Abgent, CA, USA) antibody was used as a loading control. Densitometry was performed to quantify the protein bands using ImageJ software, and a representative image of three independent experiments is shown.
For co-immunoprecipitation, HEK293T cells were transfected with mitochondrial antiviral signaling protein (MAVS), TANK-binding kinase 1 (TBK1), or interferon regulatory transcription factor 3 (IRF3)-encoding plasmids using polyethylenimine (Sigma) in the presence or absence of CCCP. Cells were pelleted and resuspended in Pierce^® IP lysis buffer and supplemented with complete protease inhibitor cocktail (Roche).

Cells were harvested and lysed with RIPA buffer (Sigma-Aldrich) containing protease and phosphatase inhibitors (Roche, Switzerland). Protein lysates were separated on a 10–12% SDS-PAGE gel, transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with 5% (w/v) skim milk in Tris-buffered saline (TBS) supplemented with 0.1% Tween-20 (TBS-Tw) for 1 h at room temperature. The PVDF membranes were then incubated with primary antibodies (Cell Signaling Technology, USA: total/cleaved-PARP total/cleaved-caspase-3, total/cleaved-caspase-7, cleaved-caspase-8, total/cleaved-caspase-9, total/phosphorylated-eIF2α; Santa Cruz Biotechnology: G3BP1, TIA-1, GFP, and eIF3η) at 4°C overnight, followed by HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies for 1 h at room temperature. Tubulin or β-actin (Abgent, USA) antibody was used as a loading control. The membranes were incubated with Pierce ECL Western Blotting Substrate (ECL Solution Kit; Thermo Fisher Scientific). Band signals were displayed in a Fusion Solo Imaging System (Vilber Lourmat, France). Band intensity measurement was performed by ImageJ software.