Article Figures & Data
Figures
- FIGURE 1.
Establishment of TLR1-deficient mice. A, A mouse TLR1 genomic locus and the targeting vector. A filled box denotes a coding exon. Restriction enzymes: B, BamHI, E, EcoRI. B, Southern blot analysis of genomic DNA extracted from mouse tails digested with BamHI. DNA was electrophoresed, transferred to nylon membrane, and hybridized with the radiolabeled probe indicated in A. C, Northern blotting analysis of thioglycolate-elicited peritoneal macrophages. Total RNA (10 μg) was electrophoresed, transferred onto nylon membrane, and hybridized with a cDNA probe for TLR1. The same membrane was rehybridized with that for TLR2 and GAPDH.
- FIGURE 2.
Impaired TNF-α production in response to the mycobacterial 19-kDa lipoprotein in TLR1-deficient macrophages. A, C, D, and E, Peritoneal macrophages (1 × 105) prepared from wild-type and TLR1−/− mice were stimulated with increasing concentrations of 19-kDa lipoprotein purified from M. tuberculosis (A), live M. bovis BCG (C), S. Minnesota Re595 LPS (C), S. aureus PGN (D) and live M. bovis BCG (E) for 24 h. Then TNF-α concentration in the culture supernatant was measured by ELISA. Data are shown as the mean ± SD of triplicate wells and are representative of three independent experiments. B, IL-6 concentration was measured in the culture supernatant of wild-type and TLR1−/− macrophages stimulated with 1 μg/ml 19-kDa lipoprotein and 100 ng/ml LPS. Data are shown as the mean ± SD of triplicate wells.
- FIGURE 3.
Involvement of TLR1 in synthetic bacterial lipopeptide recognition. A and B, Peritoneal macrophages (1 × 105) from wild-type and TLR1−/− mice were cultured with increasing concentrations of Pam3CSK4 (A) and synthetic MALP-2 (B) for 24 h. Then the concentration of TNF-α was measured. Data are shown as the mean ± SD of triplicate wells and are representative of three independent experiments. C, HEK293 cells were transiently cotransfected with control vector, TLR1, TLR2, and TLR6 expression vectors plus pELAM-luc reporter plasmid. After 24 h, the cells were stimulated with 10 ng/ml Pam3CSK4 for 8 h, and the cell lysates were assayed for luciferase activity. D, HEK293 cells were transiently transfected with the indicated combination of expression vectors for 4.0 μg/ml Flag-TLR2 or Flag-TLR4, and 6.0 μg/ml HA-TLR1. Total amount of plasmid DNA was kept constant with 10 μg by supplementing with empty vector. Thirty-six hours after transfection, the cells were lysed, immunoprecipitated with anti-Flag or anti-HA Ab (IP), and subsequently immunoblotted with anti-Flag or anti-HA Ab (WB) as indicated.
- FIGURE 4.
Differences in the lipoylation altered TLR1 responsibility. Peritoneal macrophages (1 × 105) from wild-type, TLR1−/−, and TLR2−/− mice were stimulated with increasing concentrations of Myr3CSK4 (A), Lau3CSK4 (B), N-Pam-S-Lau2CSK4 (C), and JBT3002 (D) for 24 h. Then the concentration of TNF-α was measured. The results are shown as the mean ± SD of triplicate wells and are representative of three independent experiments.
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