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 520

5.2. Fractionation
of Cerebral Artery

Canine basilar arteries are collected, and homogenized using a
polytron in 400 mL of ice-cold homogenization buffer in 1.5-mL
plastic tube. The homogenate is then ultracentrifuged at 100,000 × g
at 4°C for 30 min. The supernatant (cytosolic fraction) is removed
and placed into new tube and kept on ice. The pellet (membrane
fraction) is resuspended in homogenization buffer containing 0.1%
Triton-X, and incubated on ice for 30 min to extract proteins from
membrane. Protein concentrations in samples of each fraction are
measured by protein assay (details of protein assay are in Sect. 1.2 ).
After protein assay, ×2 modifi ed Laemmli sample buffer (400 mL)
is added, and the sample is boiled for 5 min and then quenched in
ice-cold water. Samples are stored at −20°C until use.

Samples of each arterial fraction are examined using the same
procedure used for “identifi cation of PKC isoforms” described in
the previous section. To make a comparison between fractions, the
same amount of protein (10 mg) is run through the gel, and cyto-
sol and membrane fractions from same artery are applied to adja-
cent lanes. After SDS-PAGE and followed by Western blotting
using PKC isoform-specifi c primary antibodies, the immunoreac-
tive bands are detected on X-ray fi lm. The densities of each band
are quantifi ed by densitometric analysis using Image J software
(NIH). The quantity of PKC isoforms in each fraction is expressed
as “percentage of total PKC” calculated by following equation:
PKC in the fraction (%) = BD in the fraction/
(BD cytosol + BD membrane ).
BD: band density; BD cytosol : band density in cytosol fraction;
and BD membrane : band density in membrane fraction.
Figure 2 shows the chronological changes in the intracellular
distribution of PKC a and d isoforms in canine basilar artery in
canine “two-hemorrhage” SAH model.

5.3. SDS-PAGE/
Western Blotting
and Analysis

6. Measurement
of Protein Tyrosine
Kinase Activity

Protein tyrosine kinases are enzymes that catalyze phosphorylation
of specifi c tyrosine residues and are classifi ed into two families; (1)
membrane-spanning (receptor-type) tyrosine kinases and (2) non-
receptor type tyrosine kinases. Receptor-type PTKs are transmem-
brane single polypeptide chains, consisting of two domains; an
extracellular ligand-binding domain and an intracellular tyrosine
kinase domain. Receptor-type PTKs remain inactive until their
ligand(s) binds to the extracellular domain, which generally leads
to receptor dimerization, autophosphorylation, and increased
tyrosine kinase activity. Examples of receptor-type PTKs include
epidermal growth factor receptor (EGFR), fi broblast growth fac-
tor receptor, platelet-derived growth factor receptor, and vascular

6.1. Background