Effects of mTLR2 and mCD14 on LPS-induced JNK and NF-κB activation.

(A) Expression of the Flag-tagged mTLR2 in COS7 cells. COS7 cells were transiently transfected with the following: the vector alone, an expression plasmid of the Flag-tagged mTLR2, an expression plasmid of mCD14, or a combination of mTLR2/Flag and mCD14 expression plasmids. Flag-tagged mTLR2 expression was detected by immunoblotting with anti-Flag mAb. (B) Surface expression of mCD14 on COS7 cells. COS7 cells transiently transfected with expression plasmids as in (A) were stained with a control mAb (dotted line) or an anti-mCD14 mAb (solid line) followed by FITC-conjugated antirat IgG mAb. Flow cytometric analyses of COS7 cells transiently transfected with only vector and with Flag-tagged mTLR2 plus mCD14 are shown. A similar amount of mCD14 surface expression seen in double transfection was also observed in mCD14-transfected COS7 cells (data not shown). (C) LPS-induced JNK phosphorylation by mTLR2 and mCD14. Cytoplasmic cell extracts were prepared from COS7 cells transiently transfected as in (A), which were either left untreated or stimulated with 10 μg/mL LPS for 20 minutes. JNK phosphorylation was detected by Western blot analysis using an mAb specific for the phosphorylated form of JNK. Nonspecific 46-kd bands recognized by this mAb are indicated by a dotted arrow. The same filter was reblotted with an anti-JNK1 pAb. (D) LPS-mediated NF-κB DNA-binding activation is dependent on both mTLR2 and mCD14. Nuclear cell extracts were prepared from COS7 cells transiently transfected as in (A), which were either left untreated or stimulated with 10 μg/mL LPS for 20 minutes. EMSA was performed with a radio-labeled double-stranded probe containing a NF-κB binding site. NC represents negative control without nuclear extract.