PINK1 binds Tollip in HEK293 cells. (A) HEK293 cells were transfected for 48 hours with Myc-tagged PTEN-induced putative kinase 1 (PINK1) and/or HA-tagged Toll-interacting protein (Tollip). Immunoprecipitation (IP) of cell lysates was performed with the c-Myc antibody, followed by immunoblotting with the HA antibody, as indicated. Expression of the transiently transfected proteins was identified by immunoblotting with the indicated antibodies. *IgG heavy chain. Actin was used as a loading control. (B) HEK293 cell lysates were immunoprecipitated with the PINK1 antibody, followed by immunoblotting with the Tollip antibody. As a control, cell lysates were immunoprecipitated with preimmune IgG. PINK1 and Tollip expression in cell extracts was determined by immunoblotting with each antibody.
(C) HEK293 cells were fixed, permeabilized, and incubated for 24 hours with the PINK1or Tollip antibody. Cells were stained with TRITC-conjugated or FITC-conjugated secondary antibodies and 4′,6′-diamidino-2-phenylindole (DAPI). Immunostained cells were examined using confocal microscopy. Scale bar = 20 μm.