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. Author manuscript; available in PMC: 2015 Jun 1.
Published in final edited form as: Free Radic Biol Med. 2014 Mar 21;71:49–60. doi: 10.1016/j.freeradbiomed.2014.03.017

Figure 6.

Figure 6

DCP-Bio1 trapping of sulfenic acid indicates that LPA stimulates PTP1B and Akt2 oxidation in situ. PC3 cells were pretreated before stimulation with LPA for 30 min as described in Figure 4, or with N-acetylcysteine (NAC, 20 mM) for 30 min. To covalently trap sulfenic acid-modified cysteines, DCP-Bio1 was added to lysis buffer as described in Methods and incubated for 30 min at 4 °C and biotinylated proteins were affinity captured as described in Methods. Western blots of these affinity-captured samples were performed to detect PTP1B, Akt2, and SHP-2. Western blots of the starting lysate indicated that total protein amounts did not change during the experiment (data not shown for SHP-2). Prelabeled biotinylated AhpC was added based on protein concentrations prior to affinity capture and used as a procedural control for the biotin-based affinity capture, elution and gel loading steps.