Biochemical Assessments of Cerebral Vasospasm: Measurement of cGMP, PKC, and PTK in Cerebral Arteries - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 518

Separated proteins are electrophoretically transferred to a
nitrocellurose membrane in transfer buffer at 200 mA for 90 min.
Polyvinylidene difl uoride (PVDF) membrane also can be used for
SDS-PAGE/Western blotting instead of nitrocellulose membrane.
PVDF membrane has greater ability to retain proteins; however,
autofl uorescence can affect fl uorescent detection of proteins under
certain conditions. PVDF membranes require pretreatment with
methanol (15 min incubation at room temperature before use)
because of its hydrophobicity.
After transfer, the nitrocellulose membrane is incubated in 5%
nonfat milk dissolved in TBS for 1 h at room temperature with gen-
tle agitation for blocking. The membrane is then washed for 10 min
(three times) with TBS containing 0.1% Tween-20 (TBS-T),
followed by incubation overnight at 4°C with PKC isoform-specifi c
primary antibody (1:500 dilution in TBS containing 3% horse
serum). PKC isoform-specifi c antibodies are purchased from Sigma-
Aldrich (St. Louis, MO), which have been developed in rabbits
against synthetic peptides corresponding to the C-terminal amino
acid sequence of rat PKC isoforms; a (659-672), b1 (658-671), b2
(660-673), g (684-697), d (662-673), e (726-737), z (577-592),
and h (670-683). In control experiments, antigen peptides are incu-
bated with primary antibodies to verify the specifi city of signals.
Membrane is washed three times with TBS-T for 10 min each,
and then incubated 2 mg/mL of peroxidase conjugated goat anti-
rabbit IgG antibodies (1:500 dilution in TBS containing 3% horse
serum) for 7 h at room temperature. After washing the membrane
with TBS-T, bands are visualized by enhanced chemiluminescence
reagent (ECL, Amersham Japan, Tokyo, Japan) and exposure to
X-ray fi lm (Kodak, Rochester, NY). The obtained bands are con-
dsidered positive using the following criteria (1) appropriate molecular
weight, (2) loss of immunoreactive signals by the incubation with
antigen peptide, and (3) the position of the signals using positive
control(s). An example of Western blotting for PKC isoforms is
shown in Fig. 1 .

5. Intracellular
Distribution of PKC
Isozymes:
Implication of PKC
Isozyme Activation

As shown in Fig. 1 , several PKC isoforms are usually expressed in
an arterial tissue; e.g., a, d, z, and h isoforms in canine basilar
artery ( 19 ), a, d, e, and z isoforms in porcine coronary artery ( 22 ).
Thus, it is important to clarify which PKC isoforms are involved in
specifi c physiological/pathological phenomena, such as cerebral
vasospasm following SAH. In Sect. 3 , we described methods to
measure PKC activity using radioimmunoassay and enzyme-
immunoassay. However, these kits are not useful for measuring the
activity of specifi c PKC isoform(s) due to the usage of a general

5.1. Background