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Pathway Highlight: Which caspase substrates contribute to the morphological features associated with apoptosis?

Tue, 04/11/2017 - 09:20

Apoptosis, or programmed cell death, is controlled by a caspase signal cascade that activates downstream signals to induce the morphological changes used to differentiate apoptosis from other forms of cell death.  Novus Biologicals offers a variety of antibodies and tools to detect the different morphological indicators of cell death. 

Nuclear Fragmentation

Nuclear condensation starts at the nuclear membrane, hallmarked by the formation of a ring-like structure.  Further into apoptosis, the nucleus full fragments in a process known as “karyorrhexis”.  Primary antibody markers that bind and recognize DNA such as ICAD (inhibitor of caspase-activated DNAase) and Lamin B can be used to visualize nuclei.  Furthermore, TUNEL, or terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labeling, is a popular method of apoptotic cell detection.  However, research has shown that TUNEL assays may also detect necrotic cells; therefore use of a TUNEL assay in conjunction with the above antibody methods is encouraged.

ICAD antibody Lamin antibody
DFF45/ICAD Antibody [NBP1-77034] - Staining of HeLa cells with ICAD antibody at 5 ug/ml. Lamin B1 Antibody (119D5-F1) [NBP1-97889] - Immunohistochemistry on frozen sections of human colon showing nuclear lamina staining in epithelial and connective tissue cells

Membrane Blebbing

The actin-myosin network in the cortical cytoskeleton has been proposed as the driving force for membrane bleb formation in cells undergoing apoptosis.  The following proteins are involved in the maintenance and function of the cytoskeleton and are cleaved by caspases during apoptosis: PAK2, Gelsolin, Spectrin Beta 1 and Beta Catenin.  Through light or electron microscopy, shrinking and membrane blebbing of apoptotic cells can be observed using primary antibodies against these targets   

Specrtin beta 1 antibody
Spectrin beta 1 Antibody (4C3) [NB300-574] - Analysis of Spectrin beta-1 (green) showing staining in the cytoplasm of Hela cells (right) compared to a negative control without primary antibody (left).

Mitochondrial Protein Release

During apoptosis, mitochondria membrane permeabilization triggers the release of many proteins, including Cytochrome C and Smac/DIABLO. Antibodies against these targets can be indicative of mitochondria in the late phases of apoptosis. 

Cytochrome C antibody
Cytochrome c Antibody (7H8.2C12) [NB100-56503]

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  1. Taatjes DJ, Sobel BE, Budd RC. Morphological and cytochemical determination of cell death by apoptosis. [PMID: 18000678]
  2. Estaquier J, Vallette F, Vayssiere JL, Mignotte B. The mitochondrial pathways of apoptosis. [PMID: 22399422]
  3. Campos CB, Paim BA, Cosso RG, Castilho RF, Rottenberg H, Vercesi AE. Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane. [PMID: 16680678]

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