Fig. 6.

Truncated retinoid X receptor α (tRXRα) interacts with TRAF6 and activates the IKK-NF-κB pathway. a Lysates from HEK293T cells transfected with Flag-TRAF6 and/or increasing amounts of Myc-tRXRα were analyzed by immunoblotting. b CV-1 cells transfected with pNF-κB reporter alone or in combination with RXRα or TRAF6 plasmids were analyzed for nuclear factor (NF)-κB transactivation by reporter assay. Data are mean ± SEM, *P < 0.05, by two-way analysis of variance. c Subcellular localization of transfected Myc-tRXRα or Myc-RXRα in RAW264.7 cell was revealed by immunostaining using anti-Myc antibody. The localization of endogenous p65 was stained by anti-p65 antibody. Green, Myc; red, p65; blue, DAPI. Scale bar, 10 μm. d Subcellular localization of RXRα in macrophages of colon tumor tissues from mice treated with azoxymethane/dextran sodium sulfate for 120 days was determined by immunostaining using ΔN197 anti-RXRα antibody. Green, CD68; Red, ΔN197; Blue, DAPI. Scale bar, 5 μm. e Wild-type (WT)- and tRXRα-BMDMs treated with lipopolysaccharide (LPS; 50 ng/ml) for 15 min were analyzed for tRXRα interaction with TRAF6 by immunoprecipitation assays using anti-TRAF6 antibody. Immunoprecipitates were analyzed by immunoblotting. f, g HEK293T cells transfected with the indicated plasmids were analyzed by co-immunoprecipitation assays using anti-Myc or anti-Flag antibody. h RAW264.7 transfected with HA-Ub, Flag-TRAF6, and Myc-tRXRα plasmids were immunoprecipitated with anti-Flag antibody and analyzed for TRAF6 ubiquitination by immunoblotting. i WT- and tRXRα-BMDMs treated with or without LPS (50 ng/ml) for 15 min were immunoprecipitated with anti-TRAF6 antibody and analyzed for TRAF6 ubiquitination by immunoblotting. For immunoblotting, one of three or four similar experiments is shown