PINK1 enhances formation of the intermediate signalosome including IRAK1, TRAF6, and TAK1. (A),(B) PINK1
−/− or PINK1
+/+ mouse embryo fibroblasts were pretreated with 50 μM MG132 for 30 minutes(B) and stimulated with 50 ng/ml IL-1β for the indicated times. Cell lysates were immunoprecipitated with the transforming growth factor-β activated kinase 1 (TAK1) antibody, followed by immunoblotting with the IL-1 receptor-associated kinase 1 (IRAK1) antibody. The relative binding affinities ((endogenous IRAK1 bound/endogenous IRAK1)/endogenous TAK1 IP) of samples were quantified and denoted below the upper panel. The binding intensity in (B) was quantified and error bars indicate ± standard deviation in triplicate experiments (C). PINK1, PTEN-induced putative kinase 1; R.A., relative amounts.