Research Paper Volume 1, Issue 2 pp 254—265
Identification of Peroxiredoxin 1 as a novel interaction partner for the lifespan regulator protein p66Shc
Figure 1. Effects of phosphorylation and Pin1 binding on p66Shc. (a-c) The p66CH2CB-dependent ROS-generation is enhanced by phosphorylation of Ser36 but inhibited in the presence of Pin1. Changes in fluorescence of 10 μM H2DFFDA were recorded after addition of 20 μM p66CH2CB WT (a) or the p66CH2CB-Ser36Asp mutant simulating Ser36 phosphorylation (a-c), followed by 85 μM Na-dithionite (DN) and 50 μM CuSO4 (Cu) in the presence (b+c) or absence of Pin1 (a-c) and/or the dipeptide Ala-Pro (c). (d) p66CH2CB-induced mitochondrial rupture is inhibited by phosphorylation of Ser36. Mitochondrial rupture was induced after addition of 7 μM CaCl2 by addition of 20 μM p66CH2CB WT or Ser36Asp and monitored photometrically. The initial sensitization with CaCl2 was omitted for clarity. (e) H2O2 oxidizes p66CH2CB. 10 μg p66CH2CB were incubated with 0.005 % H2O2 and subjected to non-reducing SDS-PAGE