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RT-PCR Primers for the Study of Apoptosis

structure, the Fas membrane protein was identified as a member of the tumor necrosis factor (TNF) and nerve growth factor (NGF) receptor family. When the Fas ligand binds to Fas, the death pathway of the target cell is initiated.3 Cases of both up-regulation and down-regulation of the Fas system have been identified; such regulation characterizes Fas-mediated diseases. In certain animal disease models, such as the mouse spontaneous mutations lpr (lymphoproliferation), the expression of Fas is decreased. A similar observation is also seen in human Systemic Lupus Erythematosus (SLE). In various cell lines, the expression of the Fas protein is up-regulated by interferon-g (IFN-g).

Fas-mediated apoptosis requires the activation of a class of cysteine proteases, which include the ICE family.4 In humans, the ICE family includes CPP-32 and LAP-3 proteases. Triggering of Fas by either the Fas ligand or the anti-Fas antibody rapidly stimulates the proteolytic activity of ICE proteins. Overexpression of the ICE proteins strongly potentiates Fas-mediated cell death; inhibitors of ICE proteins inhibit the apoptotic process. Thus, determining the expression of ICE family proteins is an important marker for monitoring the cell death process.

During normal cell growth stages, the family of Bcl-2 proteins is present as the antagonists of cell death. When the death signal is presented to the cell, the expression of Bcl proteins is down-regulated, which helps the death signal to trigger the activation of the ICE protease family. Consequently, the cell undergoes a characteristic apoptosis process that includes endonuclease activation, cell surface alteration, and cytoskeletal reorganization. Eventually, the cell undergoes phagocytosis. In apoptosis, the dying cell is eliminated although the inflammatory response is not induced.

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This unique buffer provides a gentle method for removal of primary and secondary antibodies to allow several reprobings on the same membrane. Gentle buffer will not alter the antigen for subsequent i...
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