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. 2010 Mar 29;285(22):16830–16843. doi: 10.1074/jbc.M110.106187

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — AP-1 (Fra-1/c-Jun) mediates 15(S)-HETE-induced MMP-2 expression and activity. A, quiescent HDMVECs were treated with and without 15(S)-HETE (0.1 μm) for the indicated time periods, and cell extracts were prepared and analyzed by Western blotting for c-Fos, Fra-1, c-Jun, Jun-B, and β-tubulin levels using their respective antibodies. B, HDMVECs were transduced with Ad-GFP, Ad-dnRac1, Ad-dnMEK1, or Ad-dnJNK1 at 40 m.o.i., quiesced, treated with and without 15(S)-HETE (0.1 μm) for 1 h, and cell extracts were prepared and analyzed for either c-Fos, Fra-1, c-Jun, Jun-B, and β-tubulin levels or Fra-1 and c-Jun levels as described in A. The c-Jun blot was reprobed sequentially with anti-Rac1, anti-MEK1, or anti-JNK1 antibodies to show the overexpression of dnRac1, dnMEK1, and dnJNK1, respectively. C, HDMVECs were transfected with scrambled, Fra-1, or c-Jun siRNA, and 48 h later cell extracts were prepared and analyzed by Western blotting for Fra-1 and c-Jun levels using their specific antibodies. D and E, HDMVECs were transfected with scrambled, Fra-1, or c-Jun siRNA, quiesced, treated with and without 15(S)-HETE (0.1 μm) for 8 h, and either total cellular RNA was isolated and analyzed for MMP-2 and β-actin mRNA levels by QRT-PCR (D) or medium was collected and analyzed for MMP-2 activity by gelatin zymography (E). The bar graphs in A–E represent mean ± S.D. values of three independent experiments. *, p < 0.01 versus control or Ad-GFP or scrambled siRNA; ^†, p < 0.01 versus 15(S)-HETE or Ad-GFP plus 15(S)-HETE or scrambled siRNA plus 15(S)-HETE.

FIGURE 4. — AP-1 (Fra-1/c-Jun) mediates 15(S)-HETE-induced MMP-2 expression and activity. A, quiescent HDMVECs were treated with and without 15(S)-HETE (0.1 μm) for the indicated time periods, and cell extracts were prepared and analyzed by Western blotting for c-Fos, Fra-1, c-Jun, Jun-B, and β-tubulin levels using their respective antibodies. B, HDMVECs were transduced with Ad-GFP, Ad-dnRac1, Ad-dnMEK1, or Ad-dnJNK1 at 40 m.o.i., quiesced, treated with and without 15(S)-HETE (0.1 μm) for 1 h, and cell extracts were prepared and analyzed for either c-Fos, Fra-1, c-Jun, Jun-B, and β-tubulin levels or Fra-1 and c-Jun levels as described in A. The c-Jun blot was reprobed sequentially with anti-Rac1, anti-MEK1, or anti-JNK1 antibodies to show the overexpression of dnRac1, dnMEK1, and dnJNK1, respectively. C, HDMVECs were transfected with scrambled, Fra-1, or c-Jun siRNA, and 48 h later cell extracts were prepared and analyzed by Western blotting for Fra-1 and c-Jun levels using their specific antibodies. D and E, HDMVECs were transfected with scrambled, Fra-1, or c-Jun siRNA, quiesced, treated with and without 15(S)-HETE (0.1 μm) for 8 h, and either total cellular RNA was isolated and analyzed for MMP-2 and β-actin mRNA levels by QRT-PCR (D) or medium was collected and analyzed for MMP-2 activity by gelatin zymography (E). The bar graphs in A–E represent mean ± S.D. values of three independent experiments. *, p < 0.01 versus control or Ad-GFP or scrambled siRNA; ^†, p < 0.01 versus 15(S)-HETE or Ad-GFP plus 15(S)-HETE or scrambled siRNA plus 15(S)-HETE.