1. (A) Prdx1−/−MEFs and Prdx1+/+MEFs were stimulated with H2O2 as indicated.
(A) Prdx1−/−MEFs and Prdx1+/+MEFs were stimulated with H2O2 as indicated. Protein lysates were collected under argonized conditions by scraping cells into argon‐purged lysis buffer (see Materials and methods) and analysed under non‐reducing conditions on SDS–PAGE. Akt phosphorylation was detected on Ser473 and Thr308. Akt protein as loading control. (B) Prdx1−/−MEFs and Prdx1+/+MEFs protein lysates were treated as described under (A) and analysed for oxidized PTEN proteins. Actin as loading control. (C) Levels of oxidized PTEN proteins were evaluated by quantifying oxidized and reduced PTEN using a Fuji imaging system (LAS 3000) and related software (ImageGuage). Quantifications of staining intensities were obtained by analysing protein bands from the same ECL obtained film exposure. The y‐axis represents staining intensities in arbitrary units of oxidized PTEN. Curves represent data from three independent experiments. (D) Prdx1−/−MEFs and Prdx1+/+MEFs were stimulated with PDGF as indicated. Protein lysates were collected and analysed as described under (A). (E) Prdx1−/−MEFs and Prdx1+/+MEFs protein lysates were treated as described under (D) and analysed for oxidized PTEN proteins. Actin as loading control. (F) Data analysis was done as described under (C). All experiments shown are representative of at least three independent studies including two sets of MEF clones from Prdx1 littermates. (G) Serum starved MEFs were stimulated with H2O2 as indicated. Protein lysates were collected and analysed as described under (A). Akt substrates were detected by western blotting using an phospho‐Akt substrate antibody (RXRXXS/T).
Juxiang Cao et al. EMBO J. 2009;28:
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