FIG. 5.
In vitro interactions between BtfS and antiapoptotic Bcl-2 family members. [35S]methionine-labeled, in vitro-translated Myc-BtfS or Myc-Bax was combined with V5/His-E1B 19K, Bcl-2, Flag–Bcl-xL, or luciferase prepared by using the TnT T7 reticulocyte lysate system. The proteins were immunoprecipitated with anti-Myc monoclonal antibody in NETN buffer for 1.5 h followed by protein A-Sepharose for 0.5 h. Samples were washed in NETN buffer, resolved by SDS-PAGE (14% polyacrylamide), and visualized by autoradiography. In addition, 1 μl of each of translation reaction mixture was analyzed to verify that equal amounts of proteins were used in the binding assay.