TNF-α induces MK2 SUMOylation and MK2-mediated HSP27 phosphorylation. (A) HUVECs were pretreated with the p38-specific inhibitor SB203580 or vehicle for 30 minutes before being stimulated by TNF-α (10 ng/mL) for 0, 10, and 60 minutes. Cell lysates were probed by immunoblotting with various antibodies (IB). Transient HSP27 phosphorylation was observed in vehicle-treated control cells only. Long exposure of the anti–MK2 immunoblot shows 12-kDa band shifts for MK2 (46 kDa) after TNF-α stimulation in vehicle-treated but not SB203580-treated cells. (B) HUVECs were transfected with MK2 siRNA or scrambled siRNA for 48 hours, stimulated with TNF-α for 0, 10, and 60 minutes, and cell lysates probed with various antibodies (IB). The MK2 12-kDa band shift is not detected in cells treated with MK2 siRNA (siMK2). (C) HUVECs were transduced with Ad-SENP2 or Ad-LacZ (50 MOI) and treated with TNF-α and analyzed as in (A). Note reduced expression of the high-molecular-weight MK2 bands. (D) Quantification of SUMOylated MK2. The total density of the boxed areas for MK2 and the band-shift zone was determined using ImageJ Version 1.43u software, and the density ratio between the band-shift area versus the MK2 area for each sample lane was determined. For standardization, each ratio was divided by the ratio of the Ad-LacZ control at time 0. For (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.