The ability of all of the TLR2 mutants to recruit MyD88.

<p>HEK293T cells were co-transfected with empty vector pFLAG-CMV8, FLAG-tagged wild-type TLR2 or each of the TLR2 mutants, along with MyD88-HA. At 24 h post-transfection, the cells were stimulated with LTA, Pam3CSK4 or FSL for 30 min. The cells were lysed, and the extracts were immunoprecipitated by anti-FLAG conjugated beads. TLR2 and MyD88 were subsequently detected in the immunoprecipitated proteins by western blot with an anti-FLAG antibody and an anti-HA polyclonal antibody. The quantity of MyD88 in the whole cell lysates was also detected with the anti-HA polyclonal antibody. The experiment was repeated for three times, and one was represented.</p

Electrostatic charge and conformation of the BB and CD loop were compared between wild-type TLR2 and its mutants.

<p>A, the conformation of the BB and CD loop in wild-type TLR2, TLR2 (L663E), TLR2 (P681A) and TLR2 (N688A) was compared, and the mutated residues L663E, P681A and N688A were stick represented. B, comparison of the electrostatic surfaces of wild-type TLR2, TLR2 (L663E), TLR2 (P681A) and TLR2 (N688A), the change of positive charge (<i>blue</i>) and negative charge (<i>red</i>) in the BB loop was indicated by a circle, and the mutated residues L663E, P681A and N688A were indicated by an arrow.</p

Responsiveness of human TLR2 (P681A) to TLR2/2, TLR2/1 and TLR2/6 agonists.

<p>HEK293T cells in 24-well plates were transfected with TLR2 (P681A), TK-RL and pBIIx-luc. At 24 h post-transfection, the cells were stimulated with the indicated concentrations of LTA (A), Pam3CSK4 (B) and FSL (C) for 6 h, respectively, and then both firefly and Renilla luciferase activities were determined using a dual-luciferase assay. The experiments were repeated for three times, and all of the data were expressed as the mean ± SD. *<i>P</i><0.05, ** <i>P</i><0.01, ns, not significant.</p

Conserved amino acid residues in all but not human TLR3 TIR domain and their location in the crystal structure of human TLR2 TIR.

<p>A, All of the TIR domains, including human TLR2, TLR3, TLR1, TLR6, TLR4, TLR5, TLR7, TLR8,TLR9 and TLR10, were aligned. The sequences responsible for the structure of αA, βA, αB, and the amino acid residues specific for human TLR3 TIR and one (657 E) specific for TLR3, but semi-conserved in all other human TLRs TIRs were underlined. The BB loop was indicated as the sequence between βA and αB, and three conserved amino acid residues in all of the TLRs, except for the TLR3 TIR domain, such as L, P and N, were indicated as“*”. The numbers indicate the positions of the amino acid residues in human TLR2. B, Stick representation of all three conserved amino acid Leu (663), Pro (681) and Asn (688), and highlighting BB loop (<i>pink</i>) in the cystal structure of human TLR2 TIR domain.</p

Expression and intracellular distribution of the human TLR2 mutants.

<p>HEK293T cells in 24-well plates were transfected with each indicated mutant and analysed 24 h later. A, the cells were stained with FITC-labelled anti-FLAG and DAPI and observed under confocal fluorescence microscopy. B, the surface expression of wild-type TLR2 (<i>solid line</i>) and each of the TLR2 mutants (<i>thin line</i>) on HEK293 was detected by FACS. C, the expression of wild-type TLR2 (<i>solid line</i>) and each of the TLR2 mutants (<i>thin line</i>) was detected following permeabilisation of the cell membrane. D, the expression of each of the TLR2 mutants was determined by western blot, with β-actin as an internal control. Each experiment was repeated for three times, and one was represented.</p

Responsiveness of human TLR2 (N657E), TLR2 (L663E) and TLR2 (N688A) to TLR2/2, TLR2/1 and TLR2/6 agonists.

<p>HEK293T cells in 24-well plates were transfected with human TLR2 (N657E), TLR2 (L663E) or TLR2 (N688A), and TK-RL and pBIIx-luc. At 24 h post-transfection, the cells were stimulated with the indicated concentrations of LTA (A), Pam3CSK4 (B) and FSL (C) for 6 h, respectively, and then both firefly and Renilla luciferase activities were determined using a dual-luciferase assay. The experiments were repeated for three times, and all of the data were expressed as the mean ± SD. * indicated as <i>p</i><0.05.</p