Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Caspases are synthesized as inactive zymogens containing a pro-domain followed by large (p20) and small subunits (p10) that are proteolytically processed in a cascade of caspase activity. Initiator caspases (including 8, 9, 10, and 12) are closely coupled to proapoptotic signals. Once activated, these caspases cleave and activate downstream effector caspases (including 3, 6, and 7), which in turn cleave cytoskeletal and nuclear proteins like PARP, alpha-fodrin, DFF, and lamin A, and induce apoptosis. Cytochrome c released from mitochondria is coupled to the activation of caspase-9, a key initiator caspase. Apoptosis induced through the extrinsic mechanisms involving death receptors in the tumor necrosis factor receptor superfamily activates caspase-8. Activated caspase-8 cleaves and activates downstream effector caspases, such as caspase-1, -3, -6, and -7. Caspase-3 is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP).Necroptosis, a regulated pathway for necrotic cell death, is triggered by a number of inflammatory signals, including cytokines in the tumor necrosis factor (TNF) family, pathogen sensors such as toll-like receptors (TLRs), and ischemic injury. Necroptosis is negatively regulated by caspase-8 mediated apoptosis in which the kinase RIP/RIPK1 is cleaved. Furthermore, necroptosis is inhibited by a small molecule inhibitor of RIP, necrostatin-1 (Nec-1). Research studies show that necroptosis contributes to a number of pathological conditions, and Nec-1 has been shown to provide neuroprotection in models such as ischemic brain injury. RIP is phosphorylated at several sites within the kinase domain that are sensitive to Nec-1, including Ser14, Ser15, Ser161, and Ser166. Phosphorylation drives association with RIP3, which is required for necroptosis. Mixed lineage kinase domain-like protein (MLKL) is a pseudokinase that was identified as downstream target of RIP3 in the necroptosis pathway. During necroptosis RIP3 is phosphorylated at Ser227, which recruits MLKL and leads to its phosphorylation at Thr357 and Ser358. Knockdown of MLKL through multiple mechanisms results in inhibition of necroptosis. While the precise mechanism for MLKL-induced necroptosis is unclear, some studies have shown that necroptosis leads to oligomerization of MLKL and translocation to the plasma membrane, where it effects membrane integrity.
Specificity:Human
Intended Use:The Apoptosis/Necroptosis Antibody Sampler BioAssay™ Kit provides an economical means of detecting markers for apoptosis and necroptosis. The kit contains enough primary antibody to perform at least two western blot experiment per anitbody.
Applications:Suitable for use in Western Blot. Other applications not tested.
Recommended Dilution:Western Blot: 1:500-1:2000Optimal dilutions to be determined by the researcher.
Storage and Stability:May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
