Figure 5.

TLR4 Y672 and Y749 are essential for optimal LPS‐inducible cytokine production. (A‐I) Tlr4^−/− BMM were reconstituted with WT mouse TLR4, the indicated TLR4 mutants or empty vector (EV). Cells were then treated with 1 ng/mL LPS (A‐G) or the indicated LPS concentrations (H‐K). (A‐D) Cells were stimulated with LPS for 4 h before total RNA was collected and assessed by RT‐qPCR for mRNA levels of Il6, Il12b, Tnf, and Ifnb1. Data (mean + SEM, n = 3) are combined from three independent experiments and are normalized to the WT TLR4 + LPS sample. (E‐G) Cells were stimulated with LPS for indicated times, after which supernatants were collected and assessed for levels of IL‐6, IL‐12p40, and TNF by ELISA. Data (mean + range, n = 3) are technical replicates from one experiment, with similar observations made in two independent experiments. (H‐J) Cells were stimulated with the indicated concentrations of LPS for 24 h and supernatants were collected and assessed for levels of IL‐6, IL‐12p40, and TNF via ELISA. Data (mean + SEM, n = 3) are combined from three independent experiments and are normalized to the WT TLR4 + LPS 10 ng/mL sample. (K) HEKBlue cells stably expressing CD14 and MD‐2 and with an integrated NF‐κB mScarlet reporter were transiently transfected with human TLR4, TLR4 Y674A, TLR4 Y674F, TLR4 Y751A, or TLR4 Y751F expression constructs, and then stimulated with LPS at the indicated concentrations for 16 h. Cells were then harvested and assessed for NF‐κB‐dependent reporter activation via flow cytometry. The mScarlet geometric mean fluorescence activity was collected from cells gated on low detectable levels of TLR4‐GFP protein and each condition was normalized to the WT TLR4 + LPS 100 ng/mL sample and represented as a percentage. Data (mean ± SEM, n = 3) are combined from three independent experiments. (A‐K) Statistical analyses were performed using a repeated measures two‐way ANOVA, followed by Bonferroni's multiple comparison test (**p < 0.01, ***p < 0.001, ****p < 0.0001).