Figure 5.

S-nitroso-PTEN accumulates in the presence of GSNO and is inactivated in a time- and dose-dependent manner. (A) BPAEC, Cos 7, or MLVEC were treated with GSNO, GSH, S-nitroso-cysteinylglycine (CGSNO), S-nitroso-N'-acetylcysteine (SNOAc), or aqueous nitric oxide (AqNO) for the times and doses stated. Some cells were treated with DTT during the final 30 minutes of GSNO treatment or were preincubated with 100 μM acivicin. Lysate from some GSNO-treated cells was treated with ultraviolet light before analysis. Total PTEN protein was then detected in the lysate samples by Western blot analysis using anti-PTEN antibody. MAPK protein levels were assessed using anti-MAPK antibody to control for protein loading. S-nitrosylated PTEN (SNO-PTEN) was isolated from whole cell lysate using the biotin switch method. SNO-PTEN protein was detected by Western blot analysis using anti-PTEN antibody. Relative protein densities of the SNO-PTEN bands were compared with that of total PTEN. The data shown are single experiments. Similar patterns of results were seen in at least two trials for each cell line. (B) MLVEC cells were treated as stated. Data shown are Western blots representing total PTEN from whole cell lysate, and SNO-PTEN isolated from the same lysate using the biotin switch method. The graph represents the ratio of SNO-PTEN: total PTEN and is representive of several experiments illustrating the same pattern of SNO-PTEN: total PTEN ratios. (C) BPAEC and MLVEC cells were treated as described above. Western blots show bands representing total PTEN and SNO-PTEN isolated from the same lysate. The graph below represents the relative PTEN phosphatase activity on phosphatidylinositol 3,4,5-trisphosphate (PIP₃). Arrows associate enzyme activity with the correlating SNO-PTEN and total PTEN bands detected by Western blot.