(A) Co-IP of endogenous DAB2 and TRAF6 from protein lysates of RAW 264.7 cells. Data are representative of at least 3 independent experiments. (B) The TRAF6-binding site consensus sequence is shown with 2 putative TRAF6-binding sites in DAB2 p96 at amino acid positions 226 and 689. Schematic of N-terminal GFP-tagged DAB2 deletion mutants. (C) TRAF6 IP with DAB2 deletion mutants in HEK^TLR2 cells (left) and HEK293T cells (right). (D) HEK^blue-mTLR2 cells were transfected with control GFP plasmid, DAB2 p96, or T6 mutant and treated with LTA (1 μg/ml) for 14 hours. Absorbance at 620 nm corresponds to NF-κB activity. The experiment was performed with 5 replicates. Data represent at least 2 independent experiments (C and D) and are expressed as the mean ± SEM. *P = 0.0276, by 1-way ANOVA with Fisher’s least significant difference test (LSD). (E) mRNA expression of Il8 and Tnfa in HEK^blue-mTLR2 cells transfected with DAB2 p96 or T6 mutant and treated with LTA (1 μg/ml) for 6 hours. The experiment was performed with 6 replicates. Data represent the mean ± SEM. *P ≤ 0.0092, by 2-tailed, unpaired Student’s t test. Data were normalized to B2m and are expressed as the fold increase compared with untreated cells. (F) RAW 264.7 cells were transfected with either DAB2 p96 or T6 mutant before stimulation with LTA for 3 and 6 hours. Expression of IL-1β or TNF-α was analyzed by flow cytometry in GFP⁺ cells. Data represent at least 2 independent experiments and are expressed as the fold change in cytokine MFI compared with nontreated cells. *P < 0.001, by 2-way repeated-measures ANOVA with Sidak’s multiple comparisons test (mean ± SD). (G) Cells obtained from BAL fluid from critically ill patients were subjected to co-IP of TRAF6 and DAB2. Anti-TRAF6 Ab or control IgG was used in each sample for IP, followed by immunoblotting (IB). The specific DAB2 band is indicated.