Fig. 5

Increased A20-TRAF6, A20-TRAF3, and Smad6-A20 interactions in bid-deficient glia. a wt and bid^−/− mixed glia were transfected with TRAF6-FLAG and stimulated for 1 h with LPS (100 ng/ml) 20 h post transfection. The cells were lysed in RIPA buffer, and anti-FLAG was immunoprecipitated from the samples. A20-TRAF6-FLAG interaction was detected by Western blotting using an anti-A20 antibody (n = 2 separate experiments, repeated with similar results). b wt and bid^−/− glia were transfected with ubiquitin-HA for 20 h and subsequently stimulated with Pam₃CSK4 (Pam, 100 ng/ml) or PolyI:C (100 ng/ml) for 1 h before being lysed in RIPA buffer. Anti-TRAF3 was co-immunoprecipitated from each of the samples, and TRAF3-A20 interactions were determined by Western blotting using anti-A20 (n = 1 experiment). c wt and bid-deficient mixed glia were transfected with Smad6-FLAG, or control vector pcDNA4.1, 20 h prior to stimulation with PolyI:C (100 ng/ml) or LPS (100 ng/ml) in full serum media. Following a 1-h stimulation, the cells were lysed in RIPA buffer, containing protease and phosphatase inhibitors, and the protein concentration was assessed using BCA assay. Anti-FLAG was immunoprecipitated from each of the samples, and the samples were prepared for Western blot analysis. The membrane was exposed to anti-A20 to determine the Smad6-FLAG-A20 interactions (n = 2 experiments, repeated with similar results).  All samples were lysed in RIPA buffer containing protease inhibitors, and protein concentrations were determined by BCA assay. Anti-IgG was used as co-immunoprecipitation and pull-down controls for each experiment. d Schematic showing the mechanism of action of Bid in response to TLR2 and − 3 and − 4 stimulation in glia and macrophages. Bid sequesters Smad6 and A20 thereby preventing Smad6-mediated recruitment of A20 to the E3 ligases, resulting in a lack of polyubiquitin chain cleavage and promotes pro-inflammatory signaling