Co-immunoprecipitation

To detect interactions with Bad and Bcl-xl, a co-immunoprecipitation assay was performed. Briefly, cells were collected and lysed with cold RIPA lysis buffer (Cell Signaling Technology, Danvers, MA) for 15 min at 4°C. The supernatant was collected, to which primary Bad antibody was added (Cell Signaling Technology). After overnight incubation, protein A/G plus-Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added and shaken on a horizontal shaker for 1 h at 4°C. Immunoprecipitated pellets were then collected and washed with cold RIPA lysis buffer. Finally, the immunoprecipitated pellets were mixed with the sodium dodecyl sulfate polyacrylamide loading buffer and boiled to remove the beads.