<p>(A) L929 cells were treated with TNF in the presence of 20 µM Nec-1 or 50 µM of the Erk inhibitor U0126. Cell death was determined by MTS assay. (B) The effect of Nec-1 on Erk or Jnk phosphorylation. L929 cells treated with 10 ng/ml of mTNF in the presence or absence of 30 µM Nec-1 were analyzed for Erk activation (p-p54 and p-p46) or Jnk activation by Western blot. (C) Erk inhibitor U0126 and Nec-1 blocks TNF-induced necrosis in Jurkat cells. Jurkat 4E3 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023209#pone.0023209-Chan2" target="_blank">[20]</a> were treated with Erk inhibitor (50 µM U0126), Jnk inhibitor (30 µM Jnk inhibitor II), p38 inhibitor (50 µM SB203580), or 10 µM Nec-1 and stimulated with 100 ng/ml recombinant human TNF plus 10 µM zVAD-fmk for 14 hours. Cell death was determined by MTS assay. (D) Jurkat 4E3 cells were stimulated with 10 ng/ml PMA for 10 minutes in the presence of 30 µM Nec-1 or 50 µM U0126. Phospho-Erk was examined by Western blot as indicated. (E) Jurkat 4E3 cells were treated with the indicated doses of Nec-1 and 100 nM calyculin A. The expression of the two different PKA isoforms and phosphorylation of protein kinase A substrates was determined by Western blot with specific μantibodies as described in materials and methods.</p
