Article Figures & Data
Figures
- FIGURE 1.
TLR9 is retained intracellularly, TLR4 traffics to the cell surface. A and B, Flow cytometric analyses. A, Ramos B cells were stained directly with an anti-TLR9 mAb (Surface) or were fixed and permeabilized before staining (Total). Shaded histograms represent cells stained with an isotype-matched control mAb. B, HEK293T cells transfected with TLR4-GFP (upper panels) or TLR9-GFP (lower panels) were stained for surface or total expression, as in A, using PE-conjugated anti-TLR4 or anti-TLR9 mAbs. C, LSC microscopy analysis. Live HEK293T cells transfected with TLR4-YFP (left panels) or TLR9-YFP (right panels) were treated with 100 μg/ml cycloheximide for 1 h before visualization to block protein synthesis. A single section (0.8 μm) is shown for YFP (upper panels) and as an overlay of YFP with the differential interference contrast image (lower panels). Scalebar, 4.5 μm.
- FIGURE 2.
TLR9 colocalizes with ER markers. HeLa cells were cotransfected with TLR9-YFP and either Rab5-CFP (early endosome), Rab7-CFP (late endosome), or Rab11-CFP (recycling endosome); with TLR9-GFP and CD63-mRFP (lysosome); and with TLR9-CFP and pEYFP-ER (a commercial marker for ER). Cells were fixed, mounted, and examined by LSC microscopy. Single 0.8-μm sections pseudocolored green (TLR9, left panels) and red (organelle marker, middle panels) are shown. An overlay of the two channels is shown in the right panels. Scalebar, 9 μm.
- FIGURE 3.
Stably expressed TLR9 resides in the ER. A, HEK293 cells stably expressing HA-TLR9 were stained for TLR9 (with anti-HA-Alexa 488) and the ER markers, heat shock protein 90 (upper panels, two connected cells) and KDEL (lower panels), using Alexa 546 secondary Abs. Single 0.8-μm sections from LSC microscopy are shown for TLR9 (left panels) and ER markers (middle panels). Overlays are shown in the right panels. B and C, TLR9 does not enter the Golgi. B, HA immunoprecipitates (from the same cells as in A) were incubated with medium alone (−), EndoH (H), or PNGaseF (F). The proteins were analyzed by SDS-PAGE and immunoblotting using an anti-HA-HRP mAb. C, Untagged TLR9, labeled with [35S]methionine and chased for the indicated times, was immunoprecipiated and treated with glycosidase as in B. Shown is the autoradiograph of the SDS-PAGE gel.
- FIGURE 4.
Endogenous TLR9 is retained intracellularly in B cell lines. A, Four B cell lines (BJAB, Raji, WIL2, and JY) were permeabilized, labeled with anti-TLR9 Ab, and stained with an Alexa 488 secondary Ab. Shaded profiles represent isotype controls. B, Endogenous TLR9 from the four B cell lines was immunoprecipated and analyzed by SDS-PAGE. After transferring to nitrocellulose, TLR9 was detected with the same Ab. In addition to the TLR9 band (→), a faint nonspecific band migrating immediately above TLR9 is evident in all samples.
- FIGURE 5.
Endogenous TLR9 is in the ER. A, BJAB cells were stained for calnexin, an ER marker (red), and TLR9 (green). Shown is a 0.7-μm section of TLR9 (upper left), calnexin (upper right), an overlay of the two (lower left), and the differential contrast image (lower right). Scalebar, 4.5 μm. B, TLR9 was immunoprecipitated from BJAB cells and digested with EndoH (H) or PNGaseF (F) as in Fig. 3B. The trace upper band in the EndoH digest corresponds to the nonspecific band seen in Fig. 4B.
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