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Caspase-2 processing in apoptotic cells can be detected in
apoptotic cell lysates by immunoblotting with specifi c caspase-2
antibodies. However, it is important to note that this approach is
not necessarily a measure of caspase-2 activation, since the initial
activation of caspase-2 occurs by dimerization and does not require
proteolytic processing [ 12 , 37 ]. Furthermore, in apoptotic cells,
caspase-2 zymogen can be cleaved by active caspase-3, which con-
tributes to the majority of the caspase activity present in cells
undergoing apoptosis.
A robust method to detect active caspase-2 in apoptotic cells is
the use of a biotin-tagged peptide inhibitor such as the pan-caspase
inhibitor biotin-VAD-FMK, which acts to irreversibly bind to the
active site of activated caspases [ 38 – 40 ]. Active caspase-2 can then
be immunoprecipitated using immobilized streptavidin and
detected by immunoblotting [ 41 , 42 ]. This chapter describes some
of these routinely used methods to assess caspase-2 activation,
function, and activity during apoptosis.2 Materials
- A humidifi ed incubator at 37 °C with 5 % CO 2.
- Cell culture medium: Dulbecco’s Modifi ed Eagle’s Medium
(DMEM) or RPMI (commercially available from Sigma or SAFC
2.1 Tissue Culture
of Cell Lines
Fig. 2 In vitro processing of caspase-2. [^35 S]-methionine-labeled caspase-2 was
translated in vitro and incubated with recombinant caspase-2 protein (rCasp2) or
with cell lysates from untreated or γ-irradiation (IR)-treated cells. Caspase-2
cleavage was assessed by autoradiography. Cleavage of caspase-2 by IR-treated
cell lysate yields some differential products and is likely to represent cleavage by
caspase-3, the predominant activated effector caspase in apoptotic cells.
Caspase-2 major cleavage products are seen at 37, 19, and 14/12 kDaLoretta Dorstyn and Sharad Kumarhttp://www.ebook3000.com