FIGURE 4.

Calcium signaling regulates TLR4 endocytosis. A, RAW 264.7 cells were pretreated with inhibitors for 30 min prior to stimulation with LPS. Cells were lysed and endosomes were enriched by sucrose gradient centrifugation. TLR4 and LAMP-1 levels were analyzed by Western blotting. B, RAW 264.7 cells were treated similarly to A but stimulated with biotin-conjugated LPS (Biot-LPS). Levels of TLR4 and biotin-LPS in the endosome enriched fraction were determined by using TLR4-specific antibody and streptavidin-HRP (SA-HRP), respectively. Results from one out of three experiments are shown.