Caspases,Paracaspases, and Metacaspases Methods and Protocols

131

1. Supplement egg extract from Subheading 3.1 with 1:20 dilu-
   tion of 20× EM (and any treatments you wish you to exam-
   ine). Incubate this extract at room temperature for 6 h.


2. Every 30 or 60 min, collect 20 μL of extract and apply to a
   0.1- μM fi lter tube. Centrifuge at 9,300 × g for 10 min at 4 °C.
   The fi lter will trap the mitochondria and any cytochrome c
   that has been released will be within the fl ow through.


3. Add 10 μL of SDS sample buffer ( see Subheading 2.5 ) to the
   fl ow through, and subject to SDS-PAGE gel (either 4–20 %
   gradient or 15 % gel).


4. Immunoblot with an anti-cytochrome c antibody. We recom-
   mend a 1:2,000 dilution of the primary antibody into 5 % fat-
   free milk and incubation of the membrane overnight at 4 °C.
   1–2 h incubation with the secondary antibody, at a dilution of
   1:10,000 should also be suffi cient.


5. Supplement cytosolic fraction of egg extract from
   Subheading 3.2 with 1:20 dilution of 20× EM.


6. Supplement cytosolic fractions with cytochrome c , at concen-
   trations between 0.6 and 1 ng/μL.


7. Incubate at room temperature for 120 min. Take samples at
   15, 30, 60, 90, and 120 min to assess caspase activity as
   described in Subheading 3.3.

Mitochondria play a key role in the apoptotic signaling cascade,

responsible for releasing pro-apoptotic factors such as cytochrome

c following MOMP. The BLC-2 family of proteins is the gate-

keeper of this mitochondrial permeabilization, with pro-apoptotic

BCL-2 family proteins inducing MOMP and anti-apoptotic BCL-2

family proteins inhibiting induction of MOMP. In the X. laevis egg

extract, caspase-2 activation lies upstream of mitochondria [ 13 ],

while executioner caspase-3 and caspase-7 lie downstream of mito-

chondria and cytochrome c release. An important aspect of study-

ing apoptosis and caspase activation is determining if the initiating

stimulus lies upstream or downstream of mitochondria. Using the

X. laevis egg extract system, this is easily achieved using techniques

already described in combination with the simple addition of either

a pro-apoptotic or anti-apoptotic BCL-2 family member.

An important pro-apoptotic BCL-2 family member is BID. This

protein belongs to a subfamily of BCL-2 family proteins known as

BH3-only proteins. BID is cleaved by the initiator caspases, cas-

pase- 8 and caspase-2, to form tBID. This truncated BID is the

fully active form of BID and is a potent inducer of MOMP and

cytochrome c release. This property allows it to be used as a tool to

dissect if there are key signals upstream of mitochondria and the

BCL-2 family of proteins important in inducing caspase activation

3.6.1 Assessment
of Cytochrome c Release
from Mitochondria
in X. laevis Egg Extract

3.6.2 Induction
of Apoptosis in Cytosolic
Fractions of X. laevis
Egg Extract Using
Cytochrome c

3.7 Assessing
the Contribution
of a Pre- mitochondrial
Signal to Caspase
Activation

3.7.1 Using Recombinant
tBID to Determine if
Inhibition of Caspase
Activation Lies Upstream
of Mitochondria and BCL-2
Family Proteins

Methods for the Study of Caspase Activation in the Xenopus laevis Oocyte...