p38MAPK (p38) increases mitochondrial ROS levels via MK2. (A) WT and MK2-/- MEFs were pretreated with vehicle or BIRB796 (B-796) (50 nM) for 1 hour and then subjected to HR: hypoxia (6 hours) and reoxygenation (15 min). Expression of phosphorylated and non-phosphorylated p38MAPK, MK2 and HSP25 was determined. (B, C) For mitochondrial ROS measurements WT and MK2-/- MEFs were pretreated with either vehicle, BIRB796 (B-796) (50 nM) or N-acetyl cysteine (NAC) (7.5 mM) for 1 hour and exposed to the HR protocol followed by ROS measurement as described in Methods. (D-F) 72 hours after transfection with MK2 siRNAs (500 nM) or control siRNAs (500 nM), HL-1 cells were subjected to HR: hypoxia (6 hours) and reoxygenation (15 min) and analyzed for the effect of MK2 knockdown on p38MAPK/MK2 signaling (D), and mitochondrial ROS production (E, F) during HR as described in the Methods. Representative immunoblots (A, D), fluorescence images (B, E) and summary graphs (C, F) are shown. The data are expressed as mean ± SEM (n = 6-8). **p < 0.01, ***p < 0.001 vs. WT MEFs undergoing HR; #p < 0.001 vs. control siRNAs transfected HL-1 cells, subjected to HR.