Figure 3.

GRP78 down-regulates surface levels of TLR4 on dendritic cells. (A) DC2.4 cells were treated with AF488-GRP78 or BSA (1 µM) at 4°C for 1 h, and then TLR4 was stained using anti-TLR4 and AF555-conjugated (red) goat anti-mouse IgG. (B) Immunoblot analyses of the interaction of TLR4 with GST-GRP78, after incubation with membrane lysates of DC2.4 cells, followed by precipitation by glutathione agarose beads. (C) Enzyme-linked immunosorbent assay of TNF-α in wild-type or TLR4KO bone marrow-derived dendritic cells stimulated for 4 h by LPS together with GRP78 at the concentrations indicated. (D) DC2.4 cells were incubated with GRP78 at 37°C for 30 min, surface TLR4 was stained and detected by confocal laser scanning microscopy (CLSM). (E) DC2.4 cells were treated with GRP78 for 30 min. TLR4 surface staining was measured by FCM. Dot plots of FCM data (left) and percentage of mean fluorescence intensity (MFI, right) are depicted. Percentage of surface TLR4 (MFI) were analyzed using the following formula: (GRP78 treatment − isotype)/(NC − isotype). (F) DC2.4 cells were incubated with GRP78 at 37°C for 1 h, surface TLR4 (green) on non-permeabilized cells and intracellular TLR4 and LAPM1 (red) on permeabilized cells were stained and detected by CLSM. Error bars represent mean ± SD from triplicate samples in one experiment. All images for all panels are representative of at least three independent experiments in which >100 cells were examined and >95% of cells showed similar staining. Scale bar, 20 µm.