Detection of JUP in plaques by immunohistochemistry and in secretome by immunoblotting.

a) Overview of JUP immunoreactivity on endarterectomised tissue. Strong staining in the atherosclerotic plaque tissue is observed. b) Clockwise from top left (400-fold magnification): H&E staining (1), anti-CD68 (2), negative control (3), and anti-JUP staining (4). c and d) Six plaque secretomes (lanes 1–6), two control secretomes (lanes 7 and 8) and GST-tagged JUP (lane 9, 107 kD) were immunoblotted with anti-JUP mAb 2C9 (c) and scFv 25G5 (d). e) Competition experiment with mAb 2G9 (which replaced 2C9). Western blots containing recombinant GST-tagged JUP (lane 1, 107 kD), ACS plasma (lane 2), and secretome (4.5 µl in lane 3 and 1.9 µl in lane 4) were incubated with mAb 2G9, which was (blot on the right) or was not (blot on the left) pre-incubated with soluble, recombinant GST-tagged JUP protein. f) Competition experiment with scFv 25G5. Western blots containing different amount of atherosclerotic plaque secretome (lane 1∶4.5 µl, lane 2∶1.9 µl, lane 3∶0.9 µl, lane 4∶0.4 µl) were incubated with scFv 25G5, which was (blot on the right) or was not (blot on the left) pre-incubated with soluble, recombinant GST-tagged JUP protein. For all immunoblots: known molecular weights of protein markers are depicted on the left and estimated molecular weights of detected protein bands are depicted on the right of both Western blots.