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. 2003 Sep 15;162(6):1099–1110. doi: 10.1083/jcb.200304093

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Copyright © 2003, The Rockefeller University Press

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Figure 4. — TLR2 and TLR1 mutants inhibit NFκB activation in HEK 293 cells in stimulation with araLAM and zymosan. (A) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by a single point mutated TLR2 construct, TLR2-P681H. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-P681H (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. Luciferase activity is expressed in normalized RLU as the ratio of NFκB-dependent firefly luciferase activity to NFκB-independent renilla luciferase activity. Data shown are the mean ± SD of triplicate wells. (B) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a TLR2 mutant missing the TIR domain TLR2-ΔTIR. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-ΔTIR (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (C) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a cytoplasmic deletion mutant of TLR1, TLR1-Δcyt. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR1-Δcyt (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control.