Supplemental Material

This article contains the following supporting material:

• Supplemental Figures S1-S5 - Figure legend for supplement data
  
  S1. Migration retardation ofp66shcA and p66shcA(S36A) in response to H2O2. p66shcA and p66shcA(536A) (both myc-tagged) were overexpressed in NIH3T3 cells, which were serum starved overnight and stimulated with 0.1 or 0.4 mM H2O2 for 10 minutes. The p66shc proteins were detected by western blot using anti-myc antibodies. Three conclusions were drawn. 1) migration retardation is difficult to detect for transfected ShcA proteins; 2) p66ShcA and p66shcA(S36A) run differently even at basal level; 3). There is still some shift for p66shcA(S36A) in response to 0.4 mM of H2O2 although the shift is less obvious than p66shcA.
  
  S2. H202 treatment led to translocation of FOXO3a from the nucleus to the cytoplasm. Cells were treated with H2O2 for different periods of time, harvested, and separated to the nuclear and cytoplasmic fractions. The levels of FOXO3a, TopBP1 (nucleus control), and p66shcA (cytoplasm control) were determined by western blot. Note that four fold more total proteins were loaded for the cytoplasmic fractions.
  
  S3. H2O2 treatment led to rapid down-regulation of p27. Cells were treated with 0.4mM of H2O2 for different periods of time and the levels of p27 were determined by Western blot with actin as a control.
  
  S4. Normal Ser36 phosphorylation in p66shcA (Y3F). COS7 cells expressing p66shcA or p66shcA (Y3F) were serum starved overnight and then stimulated with 0.5 mM of H2O2 for 10 minutes. p66shcA and p66shcA(Y3F) were immunoprecipitated with anti-myc antibody and tyrosine phosphorylation and Ser36 phosphorylation were detected by Western blot analysis.
  
  S5. Normal interaction between p66shc(Y3F) with p53shc in a co-IF experiments. The experiments were carried out exactly as in Fig. 8A.