Research Paper Volume 4, Issue 4 pp 290—304

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.