FIGURE 4.

Heparin-induced DUSP1 is critical for effects on JNK and p38 activity. A, BAOECs were treated with 200 μg/ml heparin for various times and harvested for Western blotting. The blot is representative of at least five similar experiments. B, HBMECs were grown on coverslips, treated with 200 μg/ml heparin for the times noted, and stained for DUSP1, and then nuclear DUSP1 levels were determined for at least 200 cells/point. DUSP1 levels are plotted relative to control samples set at 100. †, p < 0.05; **, p < 0.001. C–F, BAOECs were transfected with DUSP1 siRNA (light columns) or scrambled siRNA (dark columns) and seeded on coverslips as noted under “Experimental Procedures.” Some coverslips were stained for DUSP1 to confirm knockdown (e.g. C and D are representative of more than 200 cells/treatment, scale bars = 20 μm). After 24 h, cells were treated with TNFα (25 ng/ml) with or without a 20 min pretreatment of 200 μg/ml heparin, fixed, and stained for pJNK (E) or pp38 (F). Nuclear levels of the activated enzymes were determined. *, p < 0.01; **, p < 0.001; −, not significant.