Expression and function of the P680L mutant TLR3 allele. (A) RT-qPCR for IFNB and IFNL1 mRNA expression without stimulation (NS) or after 2 and 4 h of stimulation with 25 µg/ml poly(I:C) in P2.1 cells not transfected (NT) or stably transfected with empty vector, HA-tagged TLR3 WT, P554S, or P680L. VSV M51R, at a MOI of 1, was used as a positive stimulus for IFN induction via the TLR3-independent pathways. Mean values ± SD were calculated from two independent experiments, with biological duplicates in each experiment. (B) TLR3 mRNA levels were determined by RT-qPCR in P2.1 TLR3-deficient fibrosarcoma cells with or without transfection with empty vector, HA-tagged TLR3 WT, P554S, or P680L. Mean values ± SD were calculated from two independent experiments, with biological duplicates in each experiment. (C) TLR3 was detected on immunoblots. P2.1 cells not transfected or stably transfected with empty vector, HA-tagged TLR3 WT, or P680L were subjected to immunoprecipitation (IP) with anti-TLR3 antibody, and the immunoprecipitated protein was then immunoblotted (IB) with C-ter (C) HA antibody or N-ter (N) TLR3 antibody. GAPDH was used as a loading control for immunoblotting. Reproducible result from six independent experiments is shown. (D–F) Immunofluorescence imaging of P2.1 cells stably expressing HA-tagged WT or P680L. Intracellular distribution was assessed by colocalization (Coloc) with a subcellular marker: anti-PDI antibody for the ER, anti-EEA1 antibody for early endosomes, and anti-LAMP1 antibody for lysosomes. Cells were let un-treated (E) or incubated with 25 µg/ml poly(I:C) for 30 min (F). The images were analyzed with Imaris Coloc software, and plots were generated. About 200 cells were used for each analysis. Mean values ± SD were calculated from two independent experiments. *, P < 0.05; ****, P < 0.0001.