Figure 4.

TLR4–lipid raft complex formation is reduced by sulfatide. (A) Raw264.7 cells were treated with 200 μM of biotinylated sulfatide, with or without 1 μg/mL of LPS for 8 min. Biotinylated sulfatide was stained with streptavidin-Alexa Fluor 488 and TLR4 with anti-rabbit-Alexa Fluor 549, and the cells were prepared for confocal microscopy as described in the Methods section. (Scale bar : 10 μm) (B) Raw 264.7 cells were treated with vehicle control or 40 μM of sulfatide for 10 min, before 8 min of vehicle control or 200 ng/mL of LPS treatment. (C) Raw 264.7 cells were identically treated as those in (B), and obtained samples were immunoblotted for TLR4 and caveolin 1. All membranes were immunoblotted under identical medical X-ray film for accurate comparison. (SE, Short Exposure; LE, Long Exposure).