FIGURE 4:

Endocytosed TLR4 is recycled back to the plasma membrane for replenishment, but is transported to lysosomes in stx11-knockdown cells. (A) In the presence or absence of puromycin (puro: 2 μg/ml) or brefeldin A (BFA: 2 μg/ml), J774 cells (LPS 0 h) were treated with LPS (1 μg/ml) for 1 h and then further incubated without LPS for 4 h after LPS was washed out (LPS 1 h + w/o 4 h). Cells were directly stained with anti-TLR4 antibodies, and then the fluorescence intensity of plasma membranes was quantified as described in Figure 2A. (B) Fluorescence intensity of the plasma membrane of each cell (of at least 30 cells) from A was quantified and analyzed as described in Figure 2B. Data are presented as the means ± SE of three independent experiments. (C) At 72 h after transfection with siRNAs (control or stx11 siRNA#1), cells were treated with LPS as described in A in the presence or absence of a protease inhibitor cocktail (Nacalai Tesque; LPS 1 h + w/o 4 h). Under permeabilized conditions, cells were stained with antibodies against TLR4 and LAMP-1 followed by fluorescent dye–conjugated goat anti-mouse and rat secondary antibodies, respectively. Scale bar: 10 μm.