Research Paper Volume 4, Issue 4 pp 290—304

ATM-dependent phosphorylation of SNEVhPrp19/hPso4 is involved in extending cellular life span and suppression of apoptosis


Figure 5. Phosphorylation at S149 is necessary for apoptosis resistance and partially for life span extension conferred by SNEV. (A) Normal HUVEC as well as stable HUVEC overexpressing SNEV wt, SNEV S149A or empty vector control cells were lysed and submitted to SDS PAGE. Western Blots were probed with anti-SNEV and anti-?Actin antibodies. SNEV protein expression levels are increased five-fold in SNEV wt and ten-fold in SNEV S149A HUVEC as compared to untransfected HUVEC. (B) Growth curves of HUVEC overexpressing SNEV wt, SNEV S149A and empty vector. (C) SNEV wt as well as SNEV S149A overexpression reduces the basal level of DNA damage. Upper panel: Representative pictures of Comet assay performed with empty vector, SNEV wt and S149A point mutant overexpressing HUVEC. Lower panel: Comets were classified into 5 categories depending on the percentage of the DNA content in the tail. DNA damage levels of SNEV S149A. (D) Cells were pre-incubated with 1 mM BSO for 48h, followed by treatment with 100 μg/ml Bleomycin for 24h. Apoptosis was measured by Annexin-FITC and PI staining and subsequent flow cytometric analysis. (E) Cells were incubated with 100μg/ml Cisplatin for 24h. Apoptosis was measured as in D.