Figure 4. p38 MAPK controls mTORC2 kinase activity in response to oxidative stress.

(A) HeLa cells were treated with 10 µM SB203580 for 30 min prior to H₂O₂ (1 mM, 15 min) or IGF-1 (100 ng/mL, 10 min) stimulation. mTORC2 complex was pulled down by anti-Rictor antibody and subjected to in vitro kinase assay using inactive His-Akt as the substrate (Top). The specificity and equal loading of Rictor immunoprecipitates were confirmed by immunoblotting with indicated antibodies (Middle). The whole cell lysates were also analyzed by immunoblotting with indicated antibodies (Bottom). (Arrowhead, 82 kDa) (B) Quantification of relative mTORC2 kinase activity. The results are presented as mean + SEM. (One-way ANOVA followed by Newman-Keuls post-hoc test when appropriate; * p<0.05, ** p<0.01 vs corresponding control groups, ## p<0.01 vs corresponding non-SB203580 treated groups, n = 3) (C) HeLa cells transfected with Flag-EV or dominant negative p38α along with GST-Akt were treated with H₂O₂ (1 mM, 15 min). The whole cell lysates were prepared and analyzed by immunoblotting using indicated antibodies. (*, non-specific band)