FIGURE 3:

Knockdown of stx11 inhibits the replenishment of TLR4 on the plasma membrane in LPS-stimulated macrophages. (A) At 72 h after transfection with siRNAs (control or stx11 siRNA#1), cells were treated with LPS (1 μg/ml) for 1 h (LPS 1 h). After LPS was washed out, the cells were further incubated without LPS for the indicated times (LPS 1 h + w/o 2 h or LPS 1 h + w/o 4 h). Cells were directly stained with anti-TLR4 antibodies as described in Figure 2A. (B) Fluorescence intensity of the plasma membrane of each cell (of at least 30 cells) from A was quantified using ImageJ. Each intensity value was normalized to that of control cells in the absence of LPS, defined as 100%. Data are presented as the means ± SE of three independent experiments. (C) At 54 h after the transfection of stx11 siRNA#1, cells were transfected with plasmids expressing mVenus or mVenus-stx11 and incubated for 18 h. After stimulation with LPS (1 μg/ml) for 1 h, the cells were incubated for another 4 h without LPS. TLR4 surface expression was visualized as described in A, and the cells were permeabilized and stained with anti-EGFP antibodies followed by fluorescent dye–conjugated goat anti-rabbit secondary antibodies. TLR4 surface expression was partially rescued by the expression of mVenus-stx11 but not mVenus. (D) The number of cells apparently expressing TLR4 on the plasma membrane was analyzed by microscopy. Results are expressed as the percentage of TLR4-stained cells in cells expressing the mVenus-tagged protein (10–16 cells for each experiment). Data are presented as the means ± SE of four independent experiments. Statistical analysis was performed using two-tailed, paired Student’s t tests. Scale bar: 10 μm.