91completely different from that of caspase-14, showing broad
specifi city for various caspase substrates except WEHD-AFC, the
preferred substrate of active, mature caspase-14. We propose the
following mechanism of caspase-14 activation. Synthesis of
procaspase-14 and preproKLK7 is initiated at the stratum spino-
sum (SS) and peaks at the stratum granulosum (SG). Activated
KLK7 cleaves procaspase-14 at Y^178 , generating the intermediate
form (p20/p8), which acts on procaspase-14 leading to the pro-
duction of mature form, p17/p11. The proposed mechanism of
caspase-14 activation is thus distinct from canonical mechanisms of
caspase activation mediated by apoptosome, DISC, or infl amma-
some [ 11 , 12 ].
Here we describe techniques for measurement of caspase-14
activity using synthetic substrate, purifi cation of caspase-14 from
corneocyte extract, preparation of constitutively active caspase-14
and specifi c antibody, quantifi cation of total and active caspase-14
in corneocyte extracts using ELISA, as well as methods for caspase-
14 activation and its visualization by immunohistochemistry.2 Materials
- Plate reader (Fluoroskan Ascent FL or similar).
- Glass homogenizer (e.g., Wheaton Tenbroeck Tissue Grinder,
40 ml). - Protein concentrators (e.g., Amicon Ultra, Millipore).
- Fast Desalting column HR10/10 (Amersham Biosciences) or
equivalent. - HiPrep 16/10 Q XL column.
- Mono Q column.
- Mono S cation-exchange column.
- Chromatofocusing Mono P column.
- Superdex 75 gel column.
- Spectrophotometer with UV.
11. PD-10 column (GE healthcare).
12. 96-well ELISA plate.
13. Microtome for paraffi n sections.
14. Embedder Sakura ETP (Sakura Finetek) or similar. - Ac-WEHD-7-amino-4-trifl uoromethylcoumarin (AFC; e.g.,
from BioVision). - Caspase-14 assay buffer: 1.3 M tri-sodium citrate dihydrate,
0.1 M HEPES (pH 7.5), 60 mM NaCl, 0.01 % CHAPS, and
5 mM DTT ( see Note 1 ).
2.1 Equipment
2.2 Reagents
Caspase-14 Methods