Figure S1. The tissue pelleted after homogenization, filtration and sucrose gradient ultracentrifugation of a sample of cerebral cortex consists largely of blood vessels. The bar indicates 250 μm.

Figure S2. Western blot analysis of control and AD brain tissue homogenates shows a major reactive band with an apparent molecular mass of 40 kDa, which is consistent with the predicted molecular weight of smooth muscle actin (SMA). Lane 1 shows molecular weight markers; lanes 3, 5 and 7 are from AD cases; and 2, 4 and 6 from control brains. The membrane was probed with rabbit polyclonal antibody to SMA (1 in 500) (Abcam, Cambridge, UK).

Figure S3. Cerebrovascular smooth muscle cells (CVSMCs) immunolabeled for (A) smooth muscle actin (1:100) and (B) neprilysin (1:20) (Santa Cruz Biotechnology, Tebu Bio, Cambs, UK). The CVSMCs were grown on poly-L-lysine (Sciencell, TCS Cellworks, Bucks, UK)-coated glass coverslips, fixed in 4% paraformaldehyde, washed and incubated with ice-cold 70% methanol for 5 minutes and 10% donkey serum (Millipore, Watford, UK) for 45 minutes. The primary antibodies were added and left at 4°C overnight. Secondary antibody conjugated to alexa-fluor 488 (1:500) (Invitrogen, Paisley, UK) was added for 1 hour at room temperature. Images were obtained under a ×60 objective.

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