The role of eNOS, iNOS and NFκB in upregulation and activation of ...

Ye Y, et al. Mechanisms of COX2 upregulation by atorvastatin 8
Myocardial samples of the anterior wall of the left ventricle were perfused and rinsed
with 0.05M Tris buffer, PH 7.4, containing 0.16 mg/ml heparin to remove any red blood
cells and clots. Samples were homogenized in 5-10 ml of cold buffer (0.1 M Tris-HCL,
pH 7.8 containing 1 mM EDTA) per gram tissue, centrifuged at 10,000Xg for 15 minutes
at 4 0 C, and the supernatant was collected and stored on ice. The COX activity assay kit
measures the peroxidase activity of COX, assayed colorimetrically by monitoring the
appearance of oxidized N,N,N’,N’-tetramethyl-p-phenylenediamaine (TMPD) at 590 nm.
Each myocardial sample was tested in triplicate (the first without an inhibitor; the second
with DuP-697 (0.286 µM), a specific COX2 inhibitor; and the third with Sc560 (0.314
µM), a specific COX1 inhibitor. COX1 activity was calculated as the difference between
total COX activity in the sample without an inhibitor and the sample with Sc560, and
COX2 activity as the difference between total COX activity in the sample without an
inhibitor and the sample with DuP-697.
Nuclear extraction
Myocardial samples (0.25 g) were homogenized, mixed with Buffer A Mix [Hepes (pH
7.9) 10 mM, KCl 10 mM, EDTA 10 mM, DTT 100 mM, protease inhibitor cocktail, and
IGEPAL 10%, (Sigma, St Lois, MO)], homogenized again and incubated for 15 min on
ice, and centrifuged at 850 x g for 10 min at 4 0 C. The supernatants were discharged,
Buffer A Mix was added again and the samples incubated for an additional 15 min on ice,
and centrifuged at 15,000 x g for 3 min at 4 0 C. The supernatant were discharged and the
pellets resuspended in 150 µl of Buffer B Mix [Hepes (pH 7.9) 20mM, NaCl 0.4M,
EDTA 1 mM, glycerol 10%, protease inhibitor cocktail, and IGEPAL 10%], the tubes
were shake on ice at 200 rpm for 2h, centrifuged at 15,000 x g for 5 min at 4 0 C, and the
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