Figure 4

PINK1 facilitates IRAK1 dissociation from Tollip after IL-1β stimulation. (A) 293 IL-1RI cells were transfected for 42 hours with Myc-PINK1, HA-Tollip, or Flag-IRAK1, alone or in combination, and treated for 15 minutes with 10 ng/ml IL-1β. Cell lysates were immunoprecipitated with the HA antibody, followed by immunoblot analyses with the Flag or c-Myc antibodies. The relative binding affinities were quantified – (IRAK1 bound/IRAK1 input)/Tollip IP or (PINK1 bound/PINK1 input)/Tollip IP – and denoted below the upper panel. Open arrow, increased binding of PINK1 to Tollip. Actin used as a loading control. (B) PINK1 ^−/− or PINK1 ^+/+mouse embryonic fibroblasts were treated with 50 ng/ml IL-1β for the indicated times. Cell lysates were immunoprecipitated with the Tollip antibody, and immunoblotted with the IRAK1 antibody. The relative binding affinities were quantified and denoted below the upper panel. *IgG heavy chains. Actin served as a loading control. IRAK1, IL-1 receptor-associated kinase 1; PINK1, PTEN-induced putative kinase 1; Tollip, Toll-interacting protein.