Journal: Scientific Reports
Article Title: Polysulfide Na2S4 regulates the activation of PTEN/Akt/CREB signaling and cytotoxicity mediated by 1,4-naphthoquinone through formation of sulfur adducts
Figure Lengend Snippet: Chemical modification of cellular and recombinant PTEN by 1,4-NQ and suppression of 1,4-NQ modification of cellular PTEN. ( A ) Primary mouse hepatocytes were simultaneously treated with DMSO or 1,4-NQ and 10 (upper) or 100 (lower) µM Na 2 S 4 for 30 min, then PTEN was immunoprecipitated using anti-PTEN antibody. Western blotting was performed using the indicated antibodies. Representative blots are shown from three independent experiments. ( B ) Recombinant GST-tagged human PTEN (1 μg) was incubated with 1,4-NQ (1–8 μM) at 25 °C for 1 h. The reaction mixture was then subjected to immunoblotting, with the anti-1,4-NQ antibody, and SDS-PAGE, with Coomassie Brilliant Blue staining. Representative blots are shown from three independent experiments. ( C ) Results of nanoUPLC-MS E analysis of 1,4-NQ-modified cysteine residues in GST-tagged human PTEN. Recombinant GST-tagged human PTEN (1.7 μg) was incubated with 1,4-NQ (10 μM) at 25 °C for 30 min in a total volume of 10 μL 50 mM Tris-HCl (pH 7.5). After the reaction, PTEN protein was digested with trypsin and analyzed by nanoUPLC-MS E . The corresponding MS E data are shown in Table 1 .
Article Snippet: Anti-rabbit IgG conjugated magnetic beads (100 µL, Dynabeads M-280-sheep anti-rabbit IgG) were washed three times with Tris-buffered saline and Tween 20, then incubated with anti-PTEN antibodies (5 µL, Cell Signaling Technology, #9552) at 4 °C for 3 h. The unbound antibodies were removed and the beads resuspended in 500 µL cell lysate (1 µg/µL).
Techniques: Modification, Recombinant, Immunoprecipitation, Western Blot, Incubation, SDS Page, Staining, Mass Spectrometry