Fig. 6.

TLR4-TLR6 activation is triggered by a membrane proximal signaling event initiated by CD36. (a) Effect of wild type or C-terminal domain mutants of CD36 (CD36^Ala, CD36^Y463F, CD36^C464S) on oxLDL-induced expression of an NF-κB luciferase reporter gene in TLR4-TLR6 expressing HEK293 cells. (b) Immunoblot analysis of TLR4-precipitated proteins from HEK293 cells transfected with TLR4-tagged with a fluorescent protein (YFP), TLR6 and wild type CD36 or CD36^Y463F. Cells were untreated or treated with oxLDL (50 μg/ml) for 15 min and TLR4-precipitated proteins were immunoblotted with antibody to TLR6 or TLR4 (α-GFP). (c) CD36 immunoblot of Src- or Lyn-precipitated proteins from empty vector or CD36 transfected HEK293 cells (upper panel). Lyn immunoblot of proteins precipitated by a His-tagged CD36 C-terminal peptide in the presence or absence of soluble peptide competitors corresponding to the terminal 6, 9, or 13 amino acids of CD36 (460-472). (d) Effect of kinase inhibition on oxLDL-induced expression of an NF-κB luciferase reporter gene in CD36-TLR4-TLR6 expressing HEK293 cells. Cells were treated with oxLDL (50 μg/ml, 5 h) in the presence of kinase inhibitors, [Genistein (5 μM); PP1 (10 μM); LY294002 (LY, 5 μM)]; Wortmannin (10 nM) or inactive analogs [Daidzen (5 μM); PP3 (10 μM)] and cellular luciferase levels were measured. (e) Effect of kinase inhibition on co-precipitation of CD36 with TLR4 and TLR6 in THP-1 monocytes. Immunoblot analysis of CD36-precipitated proteins from THP-1 monocytes treated with oxLDL (50 μg/ml) in the absence or presence of kinase inhibitors described in (d). Data in all experiments are representative of three separate experiments. Data in a, d are the mean ± s.d. of triplicate samples (**p≤0.005).