Figure 6. Palmitic acid and Pam₃CSK₄ induce but DHA inhibits the recruitment of MyD88 and p47^phox (subunit of NADPH oxidase 2) into lipid raft fractions.

(A) To demonstrate the separation of lipid rafts (LR) and non-lipid raft (NLR) fractions of plasma membrane, cells were lysed and fractionated by sucrose-gradient ultracentrifugation. LR fractions (fractions 2 and 3) and NLR fractions (fractions 6–8) were identified by the presence of flotillin-1 (LR marker) and transferrin receptor (NLR marker), respectively. (* due to overwhelming expression only 20% of input lysate from fractions 6–8 was subjected to SDS-PAGE and immunoblotted with anti-TLR2 antibodies). (B) To determine whether palmitic acid (C16:0) or Pam₃CSK₄ induces recruitment of the downstream signaling components of TLR2 into LR fractions, THP-1 cells were serum starved in 1.0% FBS-RPMI-1640 for 12 hours, then treated with C16:0 (150 µM) or Pam₃CSK₄ (100 ng/ml) for indicated time periods. Fractions 1–4 were immunoblotted with anti-TLR2, anti-MyD88, anti-p47^phox, and anti-flotillin-1 antibodies. (C) Serum starved THP-1 cells were incubated with DHA (10 µM) for 1 hour then treated with C16:0 (150 µM) or Pam₃CSK₄ (100 ng/ml) for 5 minutes. Cell lysate was separated by sucrose-gradient ultracentrifugation and fractions were immunoblotted.