PINK1 facilitates assembly of the IRAK1–TRAF6 complex. (A) 293 IL-1RI cells were transfected for 48 hours with Flag-IRAK1, V5-TRAF6, or Myc-PINK1, alone or in combination, and treated for 15 minuteswith 10 ng/ml IL-1β. Cells were immunoprecipitated with the Flag antibody, followed by immunoblotting with the V5 or c-Myc antibodies. (B) PINK1
−/− or PINK1
+/+mouse embryonic fibroblasts were treated with 50 μM MG132 for 30 minutes followed by 50 ng/ml IL-1β for the indicated times. Cell lysates were immunoprecipitated with the TRAF6 antibody, and immunoblotted with the IRAK1 antibody. The relative binding affinities were quantified and denoted below the upper panel: (A)(TRAF6 bound/TRAF6 input)/IRAK1 IP,or(B) (endogenous IRAK1 bound/endogenous IRAK1)/endogenous TRAF6 IP. *IgG heavy chains. Actin was used as a loading control. The binding intensity in (B) was quantified and error bars indicate ± standard deviation in triplicate experiments (C). IRAK1, IL-1 receptor-associated kinase 1; PINK1, PTEN-induced putative kinase 1;R.A., relative amounts; TRAF6, TNF receptor-associated factor 6.