Genotype and agonist responses of TLR-deficient and WT-EFs. gDNA was extracted from the TLR-deficient and wild-type EFs and amplified by PCR using primers for amplification of TLR1, TLR2, TLR4, or TLR6 DNA as described in Table S1. The position of the amplified bands was as expected for the size of the different PCR products (A). To analyze their functional characteristics, the fibroblasts were stimulated for 4 hours with 100 ng/mL Pam₃CSK₄ (a TLR1/TLR2 agonist), 100 ng/mL MALP-2 (a TLR2/TLR6 agonist), 1 μg/mL LPS (a TLR4 agonist), or 100 ng/mL mTNF-α. MCP-1 (B), ICAM-1 (C), and IL-6 (D) mRNA were quantified by qRT-PCR and expressed as relative mRNA level, which corresponds to the ratio of mRNA in cells incubated with agonists over that in cells incubated without agonists. The mRNA expression level of the samples was normalized to β-actin mRNA. Data are mean values ± SE of 3 independent experiments.