Mapping the SNO-PTEN sites by site-directed mutagenesis. (A) (Top scheme) Structural domains of PTEN: phosphatase domain, Ca-independent C2 and a PDZ binding domain at the C-terminus. (B) SNO-PTEN levels of Cys to Ala mutants as measured by biotin switch assays. Transfected N2a cells were treated with 200 μM SNOC for 30 min before cells were harvested for biochemical analysis. Data presented here is representative of 7 experiments. (C) PTEN ubiquitination as measured by IP-Western. Transfected WT or triple mutant PTEN were IPed with anti-HA antibody and immunoprobed with an antibody against ubiquitin. (D) Protein structure of the lipid phosphatase domain with three putative Cys residues labeled based on published PTEN crystal data .