MK2 SUMOylation inhibits endothelial cell alignment by shear stress. (A) HUVECs were transduced with the appropriate adenoviral constructs (Ad-LacZ, Ad-WT-MK2, Ad-DN-MK2, or Ad-K339R) for 24 hours, stimulated under steady laminar shear stress for 6 hours (24 dynes/cm²), and then harvested for immunoblotting for HSP27 phosphorylation. Note the increased HSP27 phosphorylation in cells transduced with Ad-MK2-K339R (lane 8) compared with Ad-WT-MK2 (lane 4). Once again, this effect was inhibited by Ad-DN-MK2 (lane 6). (B) Quantification of HSP27 phosphorylation data from experiments similar to the one shown in panel A. (C) HUVECs transduced under the same conditions as in panel A were stained with Alexa Fluor 488–phalloidin to visualize actin filaments and cell alignment. Note the alignment in the direction of flow of cells transduced with Ad-MK2-K339R under shear stress versus Ad-WT-MK2. Also note the lack of alignment of ECs transduced with Ad-DN-MK2 despite shear stress stimulation. MK2 expression for panel C is shown in panel A (IB: MK2). (D) Quantification of EC alignment under steady laminar shear stress. For each experiment, a total of 200 cells from randomly chosen fields were examined. We defined aligned cells as those with their long axes oriented within ± 15 degrees relative to the direction of flow, and percentages of such cells are shown. (A-D) Representative images from 1 of 3 independent experiments and quantitative data are shown (n = 3). Values are means ± SEM. *P < .05; **P < .01.