Fig. 5
The C-terminal domain of US7 and US8 is required for the function of those proteins. a Schematic representation of US7 and US8 and their C-terminal deletion mutants (US7ΔCT and US8ΔCT) and the US8 point mutant (US8-Y204/211A). b The effect of US7 C-terminal domain in the blockade of TLR3- or TLR4-mediated cxcl10 or ifnb expression. HeLa cells were transfected with empty vector, wild-type US7, or US7ΔCT and then treated with 10 μg ml−1 poly(I:C) or 5 μg ml−1 LPS for 12 h. The mRNA expression levels of cxcl10 and ifnb genes were measured by qPCR analysis. *P < 0.001, **P < 0.05 (Student’s t-test). c US7ΔCT restores TLR3 and TLR4 levels. TLR3-Myc- or TLR4-Myc-expressing HeLa cells were transfected with wild-type US7 or US7ΔCT. Lysates were immunoblotted with anti-Myc antibody. Tubulin was used as a loading control. d The involvement of US7 C-terminal domain in the ubiquitination of TLR3 or TLR4. TLR3-Myc- or TLR4-Myc-expressing HEK 293 T cells were transfected with wild-type US7 or US7ΔCTand then treated with 20 μM MG132 for 4 h. Lysates were immunoprecipitated with anti-Myc and immunoblotted with indicated antibodies. e US7 CT domain is important for downregulating the cell surface expression of TLR4. Endogenous TLR4 surface expression of US7- or US7ΔCT-expressing U937 was assessed by FACS analysis. Right graph, validation of TLR4 cell surface expression levels in US7- or US7ΔCT-expressing cells by FACS. *P < 0.001, **P < 0.05 (Student’s t-test). f Both US8ΔCT and US8-Y204/211A are not able to inhibit TLR3- or TLR4-mediated cxcl10 or ifnb expression. HeLa cells were transfected with empty vector, wild-type US8, US8ΔCT, or US8-Y204/211A and then treated with 10 μg ml−1 poly(I:C) or 5 μg ml−1 LPS for 12 h. cxcl10 and ifnb mRNA levels were measured by qPCR analysis. *P < 0.001, **P < 0.05 (Student’s t-test). g US8-Y204/211A mutant restores TLR3 and TLR4 levels. TLR3-Myc- or TLR4-Myc-expressing HeLa cells were transfected with wild-type US8, US8ΔCT or US8-Y204/211A. Lysates were immunoblotted with indicated antibodies. h US8ΔCT or US8-Y204/211A restores TLR4 surface. Endogenous TLR4 surface expression of wild-type-, US8ΔCT-, or US8-Y204/211A-expressing U937 was assessed by FACS analysis. Bottom graph, validation of TLR4 cell surface expression levels in US8-, US8ΔCT-, or US8-Y204/211A-expressing cells by FACS. *P < 0.001, **P < 0.05 (Student’s t-test). Data are representative of three independent experiments and are presented as means ± s.d. in b, e, f, and h. Source data are provided as a Source Data file
