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Fig. 2. Intracellular distribution of PKC
a
and
d
isoforms in canine basilar artery following SAH. Basilar arteries were
obtained from a “double-hemorrhage” dog SAH model. The arterial homogenate was ultracentrifuged at 100,000 ×
g
for
30 min, and supernatant was used as cytosol fraction (
C
,
opened column
), the pellet was resuspended and used as
membrane fraction (
M
,
shaded column
). The immunoreactive bands were obtained using the same procedure used for
“identifi cation of PKC isoforms” shown in Fig.
1
. The quantitative summary is expressed as a percentage of the total
amount of each PKC isoform. **
P
< 0.01 vs. control,
##
P
< 0.01 vs. day 4 before second injection in same fraction. From
Nishizawa et al. (
19
) with permission.
endothelial growth factor receptor. Nonreceptor PTKs are located
in the cytoplasm and nucleus, and function as second messengers.
Nonreceptor PTKs are divided into ten subfamilies; Src, Abl, Jak,
Ack, Csk, Fak, Fes, Frk, Tec, and Syk (
27
).
Receptor-type and nonreceptor PTKs play crucial roles in
various physiological phenomena (e.g., cell differentiation) and
pathophysiological phenomena (e.g., carcinogenesis) (
27, 28
).
In the vasculature, PTKs are involved in smooth muscle prolifera-
tion, migration, and contraction (
29, 30
), and are known to be
activated in cerebral arteries after SAH (
31-33
). In this section, we
describe a method to measure PTK activity using a commercially
available nonradioactive assay kit, which was used in our previous
study (
32
). Using canine basilar arteries from double hemorrhage
SAH model, total PTK activity was measured using Universal
Tyrosine Kinase Assay Kit (Takara Bio Inc.). It is also possible to
measure the activity of some specifi c PTK (e.g., EGFR and FAK)
using this assay system by adding immunoprecipitation protocols
(details are described in kit instructions).
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