Figure 6. DTT reactivates p38 following inhibition by oxidation.

HeLa cells grown in 35 mm wells were treated with 100 µM PGJ2 for 1 hr or 10 mM H₂O₂ for 30 min. At 20 min prior to harvest, the indicated wells were treated with 0.4 M sorbitol to activate p38. In vitro kinase assays measuring phosphorylation of ATF2 were performed. Samples were divided in two and immune complexes containing p38 treated in vitro with 10 mM DTT for 10 minutes at room temperature. Panel A. Samples prepared for the reactivation experiment showed activating phosphorylation of p38 (top) and equal expression total p38 (bottom panel) similar to Figure 2. Panel B. In vitro kinase assay showed that DTT did not activate the unstimulated p38 (Sample 1, negative control) while DTT treatment did increase the already strong activity resulting from hyperosmotic sorbitol treatment (Sample 6, positive control). Sorbitol-stimulated p38 activity was inhibited by both H₂O₂ (compare Samples 6 and 5, without DTT, p = 0.007) and PGJ2 (compare Samples 6 and 3 without DTT, p = 0.05). DTT treatment completely restored PGJ2-treated, sorbitol stimulated p38 (Sample 3, compare to sample 6) and partially restored p38 inhibited by H₂O₂ (Sample 5). The immunoblot (top) is representative of three independent experiments used for the quantification (bottom).