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 2. Phosphorylation of SNEV at serine 149 is induced by oxidative stress in a time- and dose dependent manner. (A) Upon oxidative stress treatment, the band detected by a phospho-SNEVspecific antibody (anti-pSNEV(S149)) increases, and diminishes upon phosphatase treatment. HeLa cells were treated with 1mM H2O2 for 1h or left untreated. Lysates were incubated with CIP and subjected to Western Blotting with a specific anti-phosphoSNEV(S149) antibody, that was generated by immunizing rabbits with an artificial SNEV-phosphopeptide. (B) Human diploid fibroblasts (HDF) were treated with 0, 100, 200 or 400 μM H2O2 for 1h, scraped on ice in 2x SDS loading dye and subjected to SDS PAGE. Western Blot was detected with anti-pSNEV(S149) and anti-SNEV to compare pSNEV to total SNEV levels. anti-γH2AX antibody was used as positive control for stress-dependent phosphorylation. anti-?-Actin was used as loading control. (C) HDF were treated with 100 μM H2O2 for 0, 5, 15, 30, 60 or 120 min. Lysis and Western Blot as in B.