FIGURE 3.

Loop1 is required for proteolytic processing of TLR3. A, truncation of LRR1 to the end of Loop1 from TLR3-HA results in a molecule similar in size to the CTF generated by proteolytic cleavage. The Western blot (WB) images were from lysates of HEK293T cells transfected to express WT or truncated TLR3-HA in the absence and presence of Unc93b1, as indicated above the gel images. ΔL1–12-HA, deletion of LRR 1–12 from TLR3-HA; 344-C-HA, deletion of LRR1 up to and including 343 in LRR12 from TLR3-HA. B, shown is the effects of deleting Loop1 on TLR3 proteolytic processing in HEK293T cells. ΔL1 lacks residues 336–343 that contain Loop1. The Western blot images were probed to detect the TLR3 ECD or TIR. All images in each panel were from the same exposure of the same blot, but other samples unrelated to the results were cropped. These experiments were repeated twice, with the same results. C and D, the effect of TLR3 with mutations in or near Loop1 was on the accumulation of CTF. The presence of Unc93b1 is shown by the + symbol. The amount of CTF produced from each mutant shown on the Western blots was quantified and normalized to FL TLR3. The percentage accumulation of the CTF relative to that of wild-type TLR3 (%CTF) was shown as %CTF accumulation. L1/FLAG and L1/AU1 also contained a C-terminal HA tag, and their presence was detected the 3F10 antibody. These experiments were repeated once with reproducible results.