FIGURE 9.

The half-lives of full-length TLR3 and CTF. A, shown is the half-life of TLR3 in radiolabeled pulse-chase assays. HEK293T cells expressing TLR3-HA with or without Unc93b1 were pulse-labeled with [³⁵S]methionine and [³⁵S]cysteine and then harvested after a chase period at the times in hours indicated. TLR3 was identified by immunoprecipitation (IP) with an antibody to HA tag, SDS-PAGE, and autoradiography. The signals in the autoradiogram were quantified by use of phosphorimaging. Three independent experiments yielded comparable results. B, shown is a graph quantifying the results from A. C, determination of the half-lives of TLR3, NTF, and CTF by a CHX-chase assay is shown. HEK293T cells expressing TLR3-HA with or without Unc93b1 were treated with 50 μg/ml CHX for the period of time indicated above the Western blot (WB) image. TLR3 or truncated forms of TLR3 were identified by the presence of the HA tag or an antibody that recognized the TLR3 ECD. In the blots probed to detect the TLR3 ECD, the middle panel was not treated with peptide N-glycosidase F and appeared heterogeneous due to N-linked glycans. The bottom-most panel contains the NTF treated with peptide N-glycosidase F (PNGase F) to better resolve the NTF and allowed its quantification. D, a graph quantifying the results from the CHX-chase experiment is shown. These experiments were repeated twice, and the half-lives of the TLR3 and CTF were comparable with the values shown.