Anti β actin antibody

Total proteins of mouse lung and spleen tissues were obtained using lysis buffer (Shenggong, Shanghai, China) according to the manufacturer’s instructions. Western blotting was performed as described previously [27 (link)]. Briefly, total proteins from each sample were separated using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Redmond, WA, USA). After blocking with 5% non-fat milk (wt/vol) in phosphate-buffered saline (PBS) with Tween-20 (PBST) for 1 h at 37 °C, each membrane was incubated in PBST with anti-cleaved-caspase 3 monoclonal antibodies (CST, Trask Lane Danvers, MA, USA), anti-BAX (BCL2 associated X, apoptosis regulator) monoclonal antibodies (Biolegend), or anti-β-actin antibodies (CST) overnight at 4 °C. This was followed by washing and subsequent incubation with horseradish peroxidase (HRP)-conjugated Goat Anti-Rabbit IgG secondary antibodies (Beyotime Biotechnology, Shanghai, China). All analyses were performed in duplicate. The membranes were scanned using the Azure c300 system (Azure Biosystems, Dublin, CA, USA) and the signals were quantified using ImageJ software. Target protein levels were normalized to that of β-actin.

Mouse CD43⁻ resting B cells, which were stimulated with 1 μg/ml anti-mouse IgM Ab and 100 μg/ml poly(I:C) for 72 h, were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% Nonidet P-40) in the presence of a protease inhibitor cocktail (Sigma-Aldrich). After 30 min incubation, the cells were sonicated by ultrasonication (Emerson Electric, St. Louis, MO, USA). Supernatants were collected after centrifugation at 15,000 g for 30 min and mixed with SDS sample buffer with 2-mercaptoethanol (Sigma-Aldrich). After 5 min boiling, the proteins were separated by NuPAGE 4–12% Bis-Tris gel electrophoresis (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore). Anti-IRF4 (1:1,000 dilution, BioLegend), anti-IRF5 (1:1,000 dilution, Abcam, Cambridge, MA, USA), anti-IRF8 (1:1,000 dilution, Abcam), anti-ubiquitin (1:2,000 dilution, BioLegend), and anti-β-Actin antibodies (1:4,000 dilution, BioLegend) were used to detect the specific proteins. Chemiluminescence was developed using ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and exposed to the LAS-3000 Mini Imaging System (FUJIFILM, Tokyo, Japan).

LAD2 cells were untreated or infected with VSV (MOI = 100 or 300) for 24 h. After stopping the reaction by adding ice-cold PBS, sample buffer containing SDS was directly added to the pellets, followed by brief sonication. Then, the lysates were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Tokyo, Japan). The membranes were analyzed by immunoblotting with anti-MDA5, anti-RIG-I (both from Cell Signaling Technology, Danvers, MA, USA), anti-TLR3 (Acris Antibodies, San Diego, CA, USA), anti-OAS2 (OriGene Technologies, Inc., Rockville, MD, USA) or anti-β-actin antibody (BioLegend, San Diego, CA, USA), followed by the respective HRP-conjugated secondary anti-immunoglobulin antibodies (GE Healthcare). The membranes were developed using Luminata™ Forte Western HRP substrate (Merck Millipore).