FIGURE 1.

Interactions of parkin and Bcl-2. A, GST-parkin interacts with Bcl-2 in vitro. The supernatant of E. coli crude extract containing recombinant Bcl-2 expressed by pET-21a-Bcl-2 was incubated with glutathione-agarose beads bound with GST or GST-parkin. After incubation, the beads were washed with HNTG buffer, and the bound proteins were probed with anti-Bcl-2 antibody. The lower panels show the inputs of GST, GST-parkin. B, GST-parkin interacts with Bcl-xl in vitro. Similar experiments as in A were performed using GST-parkin to pull down Bcl-xl, which is expressed by pET-21a-Bcl-xl. The lower panels show the inputs of GST, GST-parkin. C, FLAG-parkin is co-immunoprecipitated with EGFP-Bcl-2. 293 cells expressing a combination of EGFP-Bcl-2 and FLAG-parkin or FLAG were subjected to immunoprecipitation with anti-GFP antibody. Immunoprecipitants and inputs were detected with antibodies as indicated. GAPDH served as a loading control. D, endogenous parkin is co-immunoprecipitated with Bcl-2. Mouse brain lysates were subjected to immunoprecipitation with polyclonal antisera against parkin or empty rabbit sera. Immunoprecipitants and inputs were detected with antibodies to parkin and Bcl-2. The parkin and heavy chain bands are overlapped. ▴ indicates the nonspecific bands. E, similar experiments as in A were performed using GST-tagged parkin or its deletion mutants to pull down Bcl-2. Meanwhile, 293 cells expressing a combination of FLAG-Bcl-2 and EGFP-parkin or its deletion mutants were subjected to immunoprecipitation with anti-GFP antibody to further identify the binding domain of parkin with Bcl-2. IP, immunoprecipitation; IB, immunoblot.