Figure 1.

USP20 is phosphorylated on Ser333 in SMCs and endothelial cells of atherosclerotic human arteries. The peroneal arteries from amputated human legs were sectioned into atherosclerotic (“athero”) and relatively normal (“non-athero”) segments. A, serial sections were immunostained with Cy3-conjugated mouse IgG specific for smooth muscle α-actin (ACTA2, top left), Cy3-conjugated isotype control IgG (yielding no color, not shown); rabbit IgG specific for phospho-USP20(Ser333) (p-USP20), total USP20, the endothelial cell marker von Willebrand factor (vWF), or no particular protein (control IgG), and then anti-rabbit-Alexa 546 (red) and Hoechst 33342 (blue, DNA). Athero and nonathero sections were stained in parallel and imaged with identical camera settings; the FITC filter cube was used to visualize elastin autofluorescence (green). Scale bars = 100 μm. Shown are results from a single peroneal artery, representative of peroneal artery samples from five subjects. Lumen is oriented upward. B, for each artery, protein immunofluorescence intensity was normalized to DNA fluorescence within three randomly selected 20 × objective fields (encompassing ∼80% of the artery), by an observer blind to specimen identity. These ratios were then averaged for each specimen to obtain “Protein/DNA (arbitrary units)”, plotted as individual values and means ± SD for five distinct peroneal arteries. Compared with nonathero: ∗p < 0.05 (2-way ANOVA with Sidak test for multiple comparisons). IEL, internal elastic lamina; SMC, smooth muscle cell; USP20, ubiquitin-specific peptidase 20.