MK2 SUMOylation inhibits TNF-α–induced HSP27 phosphorylation and actin filament remodeling in ECs. (A-B) HUVECs were transduced with the appropriate adenoviral constructs (Ad-LacZ, Ad-WT-MK2, Ad-DN-MK2, or Ad-MK2-K339R at 50 MOI) for 24 hours, stimulated with TNF-α (10 ng/mL) for 0-60 minutes, and then harvested for immunoblotting. Note the sustained phosphorylation of HSP27 by Ad-MK2-K339R compared with Ad-WT-MK2, and that this effect was inhibited by Ad-DN-MK2. (C) Densitometry quantification of HSP27 phosphorylation data from experiments similar to the one shown in panel B. (D) HUVECs were transduced with adenoviruses under the same conditions, stimulated with TNF-α (10 ng/mL) for 6 hours, then fixed and stained with Alexa Fluor 488–phalloidin for actin filament visualization. Note the hyperelongation of ECs transduced with Ad-MK2-K339R vs Ad-WT-MK2 when stimulated with TNF-α. Also note the lack of elongation in ECs transduced with Ad-DN-MK2 despite TNF-α stimulation. (E) Quantification of cell elongation. Cells with axial ratios (long axis/short axis) larger than 3 were counted in randomly selected fields and expressed as percentages of the total cells counted. MK2 expression for panel D is shown on the right. (A-E) Representative images from 1 of 3 independent experiments and quantitative data are shown (n = 3). Values are means ± SEM. *P < .05; **P < .01.