Co-Immunoprecipitation Assays

Co-immunoprecipitation (co-IP) assays were performed to assess the protein-protein interactions between Bcl-2, Bad, and Bax. According to previously described methods (Liang et al., 2021), a 500-μg sample of total protein was first pretreated with either rabbit polyclonal anti-Bcl-2 (1:1,000; ab196495, Abcam, United Kingdom) or rabbit polyclonal anti-Bad (1 μg/ml, ab90435, Abcam). A total of 20 μL of protein A/G agarose (Sigma) was added to each sample, and the mixtures were incubated overnight at 4°C and then centrifuged for 1 min at 12,000 g. To remove nonspecifically bound proteins, the precipitates were rinsed four times with NP-40 buffer. Agarose-bound immunocomplexes were then released by resuspension in loading buffer containing a denaturing agent. IgG was used as a negative control for precipitation. The protein levels of Bcl-2, Bax, and Bad in the precipitates were then assessed by western blotting as described above and probed with antibodies against Bcl-2, Bax, and Bad.