Figure 4

HMGB1 stimulates clustering of TLR4 in the lipid raft. RAW264.7 macrophages were treated with 10 ng/mL LPS or 1 µg/mL HMGB1 for 60 min, and lysates were subjected to sucrose gradient fractionation. (A) Lysates were then drawn off in 12 separate fractions and run on SDS-PAGE before immunoblot (IB) with flotillin-1 to identify raft-enriched fractions. Fractions were then probed for TLR4. (B) RAW264.7 cells were treated with 1 µg/mL HMGB1 for 0, 15, 60 or 90 min and fixed. Immune fluorescence microscopy was performed for TLR4 (red) and the lipid raft protein GM1 (green), and colocalization was assessed (yellow). Images are representative of at least three independent experiments.