Analysis of proteome change in the injured arterial vessel.

(A) Schematic presentation of quantitative proteome analyses. The rat carotid vessels were injured with a balloon catheter, as described in the Materials and Methods. The tissue homogenates of the injured rat carotids were fractionated into three subcellular fractions, labeled with fluorescent dyes (Cy2, Cy3, and Cy5), and separated on the 2D gels. ICA, internal carotid artery; ECA, external carotid artery; CCA, common carotid artery; LM, light membrane; HM, heavy membrane. (B) Quality control of the subcellular fractionations used in the 2D-DIGE analysis. The subcellular fractionation of the tissue homogenates was performed by differential centrifugation. Lamin B, β-actin, and transferrin receptor (TfR) were used as subcellular markers for nuclei, membranes, and cytosol (S-100), respectively. A representative immunoblot is shown (n = 3). (C) Western blot analysis of protein candidates selected from 2D-DIGE analyses. The differential expression of the indicated proteins during the recovery time after injury was evaluated by immunoblotting with specific antibodies. A representative blot is shown (n = 3). The corresponding spot numbers are indicated in parallel. GS, glutamine synthetase; PDI, protein disulfide isomerase; PCNA, proliferating cell nuclear antigen; UCHL1, ubiquitin carboxy-terminal hydrolase L1; PKACA, protein kinase A alpha catalytic subunit; VDAC1, voltage-dependent anion channel-1.