1. (A) Left panel: rate of H2O2 release per minute was analysed by calculating the linear slope of data obtained in Supplementary Figure S1A.
(A) Left panel: rate of H2O2 release per minute was analysed by calculating the linear slope of data obtained in Supplementary Figure S1A. H2O2 release is expressed per 1 × 106 cells per minute. P‐values were calculated using an unpaired Student's t‐test. Rate of H2O2 release in Prdx1−/−MEFs compared with either Prdx1−/−Prdx1WTMEFs (*P=0.04) or Prdx1−/−Prdx1Cys51/172S (**P=0.001). Right panel: protein levels and oxidation status of expressed Prdx1 in MEFs used in left panel. Protein lysates were prepared as described under Materials and methods and analysed under non‐reducing conditions. Status of over‐oxidized Prdx1 was tested by using an antibody recognizing Prdx1 (Prdx1‐SO2/SO3) (Woo et al, 2003). (B) Left panel: Prdx1−/−MEFs and Prdx1+/+MEFs were infected with retroviral constructs carrying genes for Prdx1WT or Prdx1C51/172S. MEFs were further infected with retrovirus expressing either H‐RasV12 or puromycin resistance gene only (EV) and plated in duplicates in soft agar. Colonies were counted after 18–20 days. Right panel: proteins of clones used in soft agar experiment were analysed for expression levels of Prdx1 and Ras. (C) Rate of H2O2 release per minute was analysed by calculating the linear slope of MEFs used in (B, left panel). For each clone, six wells were plated and analysed. The experiment shown here is representative of three independent studies from two different sets of MEF clones obtained from Prdx1 littermates. P‐values were calculated using an unpaired Student's t‐test. *P=0.045; **P= Differences in Prdx1+/+MEFs were not statistically significant.
Juxiang Cao et al. EMBO J. 2009;28:
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