Fig. 4
US8 promotes TLR3 and TLR4 destabilization by disrupting TLR3-UNC93B1 interaction and targeting TLR4 to lysosomes. a US8 interacts with TLR3 and TLR4. HeLa cells were transfected with empty vector or HA-US8 and then treated with 100 μM chloroquine for 4 h. Lysates were immunoprecipitated with anti-TLR3 or anti-TLR4 antibody and immunoblotted with anti-TLR3, anti-TLR4, anti-HA, or anti-Tubulin antibody. *, Ig heavy chains; **, Ig light chains. b US8 co-localizes with TLR3 or TLR4. HeLa cells expressing TLR3-Myc or Flag-TLR4/MD-2-Myc were transfected with empty vector, HA-US8 or US8-GFP treated with or without chloroquine, and then stained with anti-Myc, anti-Flag or anti-HA antibody. DAPI was used as a nuclear counterstain. Scale bars, 10 μm. c US8 promotes TLR4 ubiquitination. Cells expressing TLR4-Myc with or without HA-US8 were treated with 20 μM MG132 for 4 h and lysed. Cell extracts were immunoprecipitated with anti-Myc antibodies and then analyzed by immunoblotting with indicated antibodies. d, e US8 associates with UNC93B1. HeLa cells expressing UNC93B1-GFP were transfected with empty vector or HA-US8 and immunoblotted d or stained e with indicated antibodies. Scale bars, 10 μm. *, non-specific bands. f US8 reduces UNC93B1 stability in a lysosome-dependent manner. HeLa cells expressing UNC93B1-GFP were transfected with empty vector or HA-US8 and treated with 100 μM chloroquine for 4 h. Lysates were immunoblotted with the indicated antibodies. g US8 disrupts the TLR3-UNC93B1 interaction. TLR3-Myc-expressing HeLa cells were transfected with UNC93B1-GFP and empty vector or HA-US8 and then treated with 100 μM chloroquine. Lysates were immunoprecipitated with anti-Myc and immunoblotted with anti-GFP and anti-Myc or anti-HA antibody. *, Ig heavy chains. h US8 promotes TLR3 ubiquitination. Cells expressing TLR3-Myc with or without HA-US8 were treated with 20 μM MG132 for 4 h and lysed. Cell extracts were immunoprecipitated with anti-Myc antibodies and then analyzed by immunoblotting with indicated antibodies. Data are representative of at least three independent experiments. Source data are provided as a Source Data file
