Fig. 5.

Def6 inhibits assembly of the Raptor–p62–TRAF6 trimolecular complex. a, b Naive T cells from wt and DKO mice cultured as described in Fig. 3 were treated with vehicle control or rapamycin (20 nM) for the final 24 h of the 3-day culture. The expression levels and phosphorylation status of AKT and PRAS40 a and AMPKα and Raptor b were assessed by western blotting. c Assembly of the raptor–p62–TRAF6 trimolecular complex was evaluated by western blotting after Raptor immunoprecipitation using extracts from wt and DKO T cells. As a control, Raptor antibody immunoprecipitates in the absence of extracts were analyzed along with extracts as input. d The interaction between endogenous Def6 and p62 was determined by p62 immunoprecipitation using extracts from wt T cells. e The interaction between endogenous Def6 and TRAF6 was determined by TRAF6 immunoprecipitation as in d. f, g 293T cells were cotransfected with expression vectors for Myc-Raptor and HA-p62 f or Flag-TRAF6 and HA-p62 g, with or without Def6 expression vector. HA-p62 immunoprecipitates were analyzed by western blotting with the indicated antibodies to determine the effect of Def6 on the interaction between Raptor and p62 f or p62 and TRAF6 g. Data are representative of two independent experiments. h, i 293T cells were cotransfected with HA-Def6 and either Flag-TRAF6 h or Myc-raptor i. Cell extracts were immunoprecipitated with anti-HA antibody. The interaction between Def6 and either TRAF6 h or raptor i was determined by western blotting with the indicated antibodies