Ear fractions (2.3-fold) of p53-deficient cells when compared with wild-type cells (Fig. 6E ), a phenomena fully reverted by the reconstitution with exogenous p53-flag (Fig. 6E ). Overall these outcomes suggest that p53 is straight or indirectly involved in subcellular trafficking of non-phosphorylated eEF-2 into the nucleus. It was previously reported that eEF-2 can interact together with the protein 14-3-3 [20]. We found that eEF-2 and 14-3-3 complexes are present in each the cytoplasmic and nuclear fractions in hippocampal key cultures (Fig. 7A). Provided that most reported interactions of 14-3-3 are mediated by phosphorylated Ser or Thr residues present in their target proteins, we examined no matter if phosphorylation of eEF-2 was needed for binding to 14-3-3. We treated soluble cytoplasmic and nuclear extracts prepared from hippocampal neurons with protein phosphatase 2A, and then performed IP – immunoblot analysis with eEF-2 and 14-3-3 antibodies. eEF-2 binding to 14-3-3 was nearly abolished soon after phosphatase therapy, nevertheless it was maintained when the extract was pretreated with all the phosphatase inhibitor okadaic acid (Fig. 7A). This outcome suggests that the interaction of eEF-2 with 14-3-3 is determined by the phosphorylation status of eEF-2. To study regardless of whether or not the association among eEF-2 and 14-3-3 is determined by the presence of p53, cytoplasm and nuclear extracts of wild-type and p53-deficient cells have been analyzed by IP-immunoblot assay. We identified that the eEF-2 interaction with 14-3-3 happens independently from the presence of p53 (Fig. 7B. C). To further realize the mechanism(s) that controls the subcellular localization of eEF-2, we employed Leptomycin B, an inhibitor of CRM1-dependent nuclear protein export. Immunoblot analysis showed a substantial boost of your nuclear/cytoplasm ratio of total eEF-2 in hippocampal neurons in response to Leptomycin B remedy (1.8-fold and three.7-fold for the duration of 3 hand 6 h treatments, respectively (Fig. 7D, E). Even so, the ratio of nuclear/ cytoplasm phosphorylated eEF-2 was unaffected by Leptomycin B therapy (Fig. 7F). These benefits suggest that the subcellular localization of non-phosphorylated eEF-2 is influenced by p53 and CRM1, when phosphorylated eEF-2 subcellular localization is regulated by 14.SLF 3.Nimodipine three.PMID:23626759 Overexpression of eEF-2 increases neuronal survival To decide whether or not eEF-2 over-expression in principal neurons could influence cell survival in response to oxidative stress, we analyzed cell viability and necrotic cell death in hippocampal neurons 72 h post-infection with either eEF-2 over-expressing or control lentiviruses, and after that treated for three and 24 h with rising concentrations of CH (10, 15 and 20 M). Overexpression of eEF-2 elevated cell viability ten (15 M CH) and 7 (20 M CH) at three h after CH treatment, and 14 (10 M CH) and 16 (15 MCH) at 24 h following CH remedy (Fig. 8A, B). Similarly, eEF-2 over-expression in key neurons led to a reduce of necrotic cell death assayed as LDH release of six (ten M CH), 15 (15 M CH) and 15 (20 M CH) at three h soon after therapy with CH, and 9 (ten M CH) and 21 (15 M CH) at 24 h right after treatment with CH. To ascertain the efficiency with the over-expression and how it effects eEF-2 subcellular localization in response to oxidative stress, we treated hippocampal neurons 72 h post-infection with 15 M CH or vehicle for three h, then analyzedFree Radic Biol Med. Author manuscript; available in PMC 2014 September 29.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA A.