RIP1-Independent Effects of Necrostatin-1 Figure 4. Effects of Nec-1 on Jnk, Erk and PKA-Cb. (A) L929 cells were treated with TNF in the presence of 20 mM Nec-1 or 50 mM 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 mM 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 [20] were treated with Erk inhibitor (50 mM U0126), Jnk inhibitor (30 mM Jnk inhibitor II), p38 inhibitor (50 mM SB203580), or 10 mM Nec-1 and stimulated with 100 ng/ml recombinant human TNF plus 10 mM 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 mM Nec-1 or 50 mM 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 mantibodies as described in materials and methods. doi:10.1371/journal.pone.0023209.g004 ylation of Jnk1/2 (Fig. 4B, p54 and p46, bottom two panels). While Nec-1 had no effects on Jnk activation, the reduction of Erk phosphorylation was more prominent in the presence of Nec-1 (Fig. 4B). To more definitively determine the effect of Nec-1 on Erk, we tested the effect of Nec-1 in Jurkat cells. TNF and zVAD- fmk induced necrosis in Jurkat 4E3 cells [19,20], which was effectively inhibited by the Erk inhibitor U0126 or Nec-1 (Fig. 4C). By contrast, inhibition of p38 or Jnk had no effects on TNF/ zVAD-fmk induced necrosis in Jurkat 4E3 cells (Fig. 4C). TNF stimulation did not induce detectable level of Erk phosphorylation in Jurkat 4E3 cells (data not shown). To evaluate whether Nec-1 inhibits Erk activation in Jurkat cells, we treated the cells with phorbol myristate acetate (PMA), which strongly induced Erk phosphorylation. We found that Nec-1 did not inhibit on Erk phosphorylation in Jurkat 4E3 cells under these conditions (Fig. 4D). Thus, the effect of Nec-1 on Erk phosphorylation in L929 cells is likely due to inhibition of RIP1, which is required for downstream Erk activation in certain cells [29]. A recent report indicates that Nec-1 could inhibit PKA activity in vitro [30]. Interestingly, we previously identified PKA-C1 as a PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23209