Protein samples from carotids of at least three mice per
group were snap-frozen and stored at 80°C. Protein
samples were loaded and separated by 10% SDS-polyacrylamide
gel electrophoresis, followed by transfer to
polyvinylidene difluoride membrane. The membranes
were then blotted using goat anti-cathepsin S antibody
(ab18822; Abcam) and horseradish peroxidase–conjugated
anti-goat secondary antibody. Signal was detected
with enhanced chemiluminescence reagents (Perkin Elmer
Life Science, Boston, MA). Membranes were then
stained with Amido Black for total protein content determination.
Blots were quantified using Image J software
(v1.44, NIH, Bethesda, MD) and normalized to internal
protein bands.