Fig 2: Detection of JUP isoforms in plasma from PAOD patients with atherosclerosis and in plasma from a swine model of myocardial infarction without atherosclerosis and plaque rupture.a) Western blot containing recombinant GST-tagged JUP (lane 1, 107 kD), ACS plasma (lane 2), control plasma (lane 3) and plasma from four PAOD patients (lanes 4 to 7) were detected with mAb 2G9 (which replaced 2C9). JUP-55 and JUP-30 are clearly detected besides JUP-81. b) Western blot containing recombinant GST-tagged JUP (lane 1, 107 kD), ACS plasma (lane 2), control plasma (lane 3) and plasma samples from swine before ligation (lanes 4 and 7), three hours after ligation (lanes 5 and 8) and three days after ligation (lanes 6 and 9) were detected with mAb 2G9. JUP-81 was not detected in the swine samples, whereas JUP-30 and a protein band with a slightly larger molecular weight were detected with similar intensities before and after ligation.

Fig 3: 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.

Fig 4: Schematic overview of the four different JUP isoforms.JUP-81 and JUP-63 have an identical N-terminus (the N-terminal 303 amino acids), and JUP-63 further shares its C-terminus with cytokeratin 19 (K1C19). The sequences of JUP-55 and JUP-30 are currently unknown but they contain epitopes that are shared with JUP-81 and are thought to be located in the N-terminal region of native JUP. JUP-30 lacks (at least) the N-terminal 50 amino acids of JUP-81. JUP-81, which is also referred to as gamma-catenin, is furthermore homologous to beta-catenin. Amino acids 1, 303 and 745 of JUP-81 are indicated with arrows.

Fig 5: JUP plasma levels in patients with ACS, CAD and controls.a) Detection of JUP-81 by immunoblotting with mAb 2C9. b) Semi-quantitative analysis of Western blots. JUP plasma levels were correlated to JUP in a reference plasma that was run on each blot.