Figure 2. Hydrophilic point mutations abrogate the ability of LT-IIb-B₅ to induce TLR2/1-dependent NF-κB activation.

Wild-type (WT) LT-IIb-B₅ and indicated point mutants (all at 10 μg/ml) were tested for their capacity to activate NF-κB in reporter THP-1-Blue cells in a TLR2-or TLR1-dependent way (A and B, respectively). Prior to stimulation, the cells were pretreated for 30 min with anti-TLR2 (A) or anti-TLR1 (B) (or other anti-TLR Abs, as indicated, for control purposes). Activation was determined colorimetrically by measuring the activity of NF-κB-inducible alkaline phosphatase secreted in the culture supernatants upon 24-h incubation. Pam₃CSK₄, LPS, and FSL-1 (which activate TLR2/1, TLR4, and TLR2/6, respectively) were used for monitoring the specificity of the blocking Abs. Results are presented as means ± SD (n = 3) from one of two independent sets of experiments yielding similar findings. Asterisks indicate statistically significant (p < 0.05) activation of NF-κB compared to no-agonist control and black circles show significant (p < 0.05) inhibition of activation.