Fig. 5. Binding between parkin and -tubulin in the rat brain and HEK293 cells. (A) Ultracentrifuged rat brain homogenates were incubated with or without colchicine (25 µM for 15 minutes at 4°C) and immunoprecipitated with pre-immune serum (Pre) or anti-parkin (PK) that had been pre-incubated with or without its antigenic peptide. Immunoprecipitates and 1% of the input material (In) were analyzed by western blotting (WB) with a monoclonal antibody against -tubulin. The co-immunoprecipitation of -tubulin with parkin was not affected by colchicine, and was abolished when the antibody was pre-absorbed with its antigen. (B) Ultracentrifuged rat brain homogenates treated with or without colchicine were immunoprecipitated with an irrelevant antibody (n, anti-neurabin) or -tubulin antibody (). Precipitated proteins and 1% of the input material (In) were analyzed by western blotting with antibodies against -, ß- or -tubulins, respectively. Only a very small fraction of -tubulin was co-immunoprecipitated with /ß-tubulin heterodimers. (C) Cleared lysates from HEK293 cells transfected with or without FLAG-tagged parkin were immunoprecipitated with anti-FLAG. Precipitated proteins and 3% of input material were analyzed by western blotting with anti--tubulin. The input was also blotted with anti-FLAG to detect the expression of the parkin construct. -tubulin, which was co-immunoprecipitated with parkin, migrated at 50 kDa. IgG HC, heavy chain of IgG. The experiments were repeated at least three times with similar results.