Figure 2

PTEN is S-nitrosylated by various chemical and biological NO donors in cultured neurons. (A) PTEN nitrosylation by SNOC in a dose-dependent manner as detected by biotin-switch assays: to detect S-nitrosylated cystein residues, the cysteine residues of PTEN was first masked by methylthiolation with MMTS. Nitrosothiols were then selectively reduced by ascorbate to reform free thiol group, which reacted with biotin-HPDH. In this experiment MMTS was added to serve as a positive control since all the cysteine residues in PTEN can react with biotin-HPDP. On the contrary, ascorbte in the untreated samples were used as negative control due to no reactive cysteine residues to biotin-HPDH. (B) Specificity of PTEN S-nitrosylation by DAN assay. (C) PTEN can be S-nitrosylated in cultured neurons by SNOC (200 μM, 30 min), glutamate (200 μM, 30 min), Aβ peptides (10 μM, 4 h) but not by staurosporine (STS, 200 nM, 30 min). (D) H₂O₂-induced oxidation in primary neurons with the same treatment conditions as in (C). H₂O₂ was used at 100 μM for 2 h. (E) P-Akt was detected by Western blot analysis 30 min after treatments with SNOC (200 μM), glutamate (200 μM), Aβ peptides (10 μM) in neurons. For the right panel, 10 mM DTT was added during the 30 min treatments.