BaxΔ2 is a unique isoform of the proapoptotic protein Bax that does not target mitochondria. The proapoptotic function of BaxΔ2 is through... Show moreBaxΔ2 is a unique isoform of the proapoptotic protein Bax that does not target mitochondria. The proapoptotic function of BaxΔ2 is through forming cytotoxic aggregates in the cytosol. The cytotoxicity of BaxΔ2 is known as associated with the BH3 killing domain and the C-terminus, which recruits caspase 8. BaxΔ2 proteins without C-terminal form large cytosolic protein aggregates unable to induce caspase 8-dependent cell death. Since abnormal cytosolic protein aggregates often contain complexes of proteins that involved in many diseases, we would like to purify BaxΔ2 aggregates and examine their components. In this study, we expressed GFP-tagged BaxΔ2(Δ6) in the Bax-negative HCT116 cell line and purified the aggregates via different digestion processes. We found that most aggregates were trapped into a DNA pellet after cell lysis. Digestion with DNase could release the aggregates, which were susceptible to detergent solvent. The yield of purification is very low and needed improvement. The results from Western Blot showed that, in addition to BaxΔ2 proteins, stress granule protein TIAR was also potentially in the aggregates. Identification of the components inside aggregates will help us to understand the mechanism of BaxΔ2 cytotoxicity. Show less
