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. Author manuscript; available in PMC: 2010 Apr 3.
Published in final edited form as: Circ Res. 2010 Feb 4;106(6):1092–1102. doi: 10.1161/CIRCRESAHA.109.215723

Figure 3. Localization of PAI-1 at the MEJ.

Figure 3

Immunoblots of VSMC, EC and MEJ fractions isolated from the VCCC blotted for PAI-1 and GAPDH as a loading control. Normalized quantification of protein expression for PAI-1 in each fraction is given in the adjacent histogram, n=4 (A). Immunocytochemistry of a single focal plane of a transverse section of the VCCC labeled for PAI-1 (green) and actin (red; phalloidin) demonstrate colocalized expression of both proteins regardless of the location in the Transwell pores (B). In vivo, the expression of PAI-1 on actin bridges that form between EC and VSMC (i.e. MEJs) in mesentery, cremaster and coronary microvascular beds is quantified using confocal microscopy in both C57Bl/6 and PAI-1−/− mice, n=3 mice per experimental paradigm, 5 images per mouse (C). A representative TEM image of a MEJ from a mouse coronary arteriole labeled for PAI-1 with 10 nm gold particles is shown and quantified as number of beads per micrometer squared in D, n=3 mice per experimental paradigm, 5 images per mouse. Enlargement of white box in B is shown on right. In C, arrow indicates PAI-1 labeling. In D, “L” indicates lumen. Enlargement of red box insert in D is shown on right. Bar in B is 5 µm, bar in D is 0.5 µm, * p<0.05.