FIGURE 4.

UNC93B1 translocates the differentially glycosylated TLR3 to the plasma membrane. A, HEK293T cells were transfected with TLR3 alone or with UNC93B1. Cell surface and intracellular expression of TLR3 was determined using flow cytometry. Histograms of mock-transfected cells (pcDNA3, filled areas) or cells transfected with TLR3 (solid lines) are shown. The representative data from three experiments are shown. Increase of cell population expressing plasma membrane-localized TLR3 normalized by the number of cells expressing intracellular TLR3 is shown on the right. B, HEK293T cells were transiently transfected with plasmid encoding TLR3 alone or with UNC93B1. 48 h post-transfection cell lysates were loaded onto a 5% SDS-PAGE gel. TLR3 was detected on a Western blot using anti-TLR3 antibody (left). * indicates the differentially glycosylated form of TLR3. Cell lysates were treated with peptide N-glycosidase F (PNGase; right). The representative image from three experiments is shown. C, HEK293T cells were transiently transfected with TLR3 alone or with UNC93B1. 48 h post-transfection surface proteins were biotinylated. Cells were lysed, and the biotinylated proteins were isolated, blotted, and detected using anti-TLR3 antibody. Cytoplasmic mCerulean was detected using anti-GFP antibody. * indicates differentially glycosylated form of TLR3. The representative image from three experiments is shown.