Figure 2.

P554S and E746X TLR3 alleles are loss-of-function. (A) TLR3 mRNA levels were determined by RT-qPCR in P2.1 TLR3-deficient fibrosarcoma cells not transfected (P2.1) or stably transfected with WT TLR3 (P2.1-TLR3 WT), P554S (P2.1-TLR3 P554S) or E746X (P2.1-TLR3 E746X) mutant TLR3, N284I (P2.1-TLR3 N284I) or L412F (P2.1-TLR3 L412F) TLR3 variant, or mock vector (P2.1-mock). β-Glucuronidase (GUS) was included for normalization. The results shown are representative of three independent experiments. (B) TLR3 expression, assessed by immunoblotting (IB) after immunoprecipitation (IP), in P2.1 TLR3-deficient fibrosarcoma cells not stably transfected (P2.1) or transfected with WT TLR3, P554S or E746X mutant TLR3, N284I or L412F TLR3 variant, or mock vector, with an anti-TLR3 N-terminal (N) antibody and an anti-HA C-terminal tag antibody. The experiment shown is representative of three experiments performed. TLR3 protein extracted from HEK293T cells transfected with human WT TLR3 was included as a positive control. We used β-tubulin as an internal expression control for immunoblotting. (C) IL29 (IFN-λ1) mRNA induction, without stimulation (NS) or after 4 h of stimulation with poly(I:C), assessed by RT-qPCR, in P2.1 TLR3-deficient fibrosarcoma cells not transfected (P2.1) or transfected with WT TLR3, P554S or E746X mutant TLR3, N284I or L412F TLR3 variant, or mock vector. All transfections generated stable cell lines. β-Glucuronidase was included for normalization. Mean values ± SD were calculated from two independent experiments. (D) IFN-λ production without stimulation (NS) or after 24 h of stimulation with poly(I:C), as assessed by ELISA, in P2.1 TLR3-deficient fibrosarcoma cells not transfected (P2.1) or transfected with WT TLR3, P554S or E746X mutant TLR3, N284I or L412F TLR3 variant, or mock vector. All transfections generated stable cell lines. One experiment representative of the three performed is shown. Mean values ± SD were calculated from triplicates in one experiment.