Transient expression of the Bcl-2 family member, A1-a, results in nuclear localization and resistance to staurosporine-induced apoptosis

The Bcl-2 family of proteins has been characterized by either anti-apoptoti c or pro-apoptotic activity. Insight into how Bcl-2 family members function has been gained by determining their intracellular localization. We have g enerated a monoclonal anti-Al-a antibody and used a COS-7 overexpression sy stem to study the localization of the murine anti-apoptotic Bcl-2 family me mber, Al-a, Al-a overexpressed in COS-7 cells localized to the nucleus as d etermined by subcellular fractionation and immunofluorescent microscopy, Al -a in the COS-7 nucleus bound tightly to the nuclear matrix as evidenced by resistance to treatment with DNAsel and RNAse A and sequential extraction with 1.0% Triton X-100, 0.15 M NaCl, 0.25 M HCl, 0.5 M Tris pH 7.4 and 6 M urea. HPLC analysis of Al-a, subsequent to SDS extraction, produced fractio ns that gave multiple bands when analyzed by Western blot analysis suggesti ng a propensity to form multimers, COS-7 cells transfected with A1-a were p rotected from apoptotic induction by staurosporine treatment.