Fl425

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing 1% Triton X-100, protease inhibitors and phosphatase inhibitors. Total protein lysates were quantified, and lysates containing 1 mg of total protein were subjected to coimmunoprecipitation using a rabbit polyclonal antibody raised against Smad2/3 (FL425; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Lysates were then incubated with protein A Sepharose beads for 2 hours at 4°C. Beads were then washed three times with RIPA buffer, mixed with 2× SDS loading buffer, boiled for 5 minutes and subjected to SDS-PAGE.

BMDCs were stimulated or unstimulated with exosomes for 20 or 40 min and lysed with 1% NP-40. The lysetes of BMDCs were immunoblotted with antibody to phosphorylated IRF3 (4D4G; Cell Signaling Technology) or IRF3 (FL-425; Santa Cruz Biotechnology).

Antibody against UL37 was generated by Cocalico Biologicals, Inc. Antibodies against IRF3 (FL-425), GST (Z-5), and RIG-I (H-300) were purchased from Santa Cruz Biotechnology. Antibodies against FLAG (M2; Sigma), V5 (A190-220A; Bethyl Group), CAD (A1301-374A; Bethyl Group), PPAT (15401-1-AP; Proteintech), CTPS1 (15914-1-AP; Proteintech), MAVS (5-20337; Thermo Scientific), RIG-I (SS1A; Enzo Life Sciences), p-S172 TBK-1 (D52C2; Cell Signaling), TBK-1 (3013S; Cell Signaling), p-S396 IRF3 (4D4G; Cell Signaling), and β-actin (Ab8226; Abcam) were purchased from the indicated suppliers.