Knockdown of p38MAPKα (p38α) decreases ROS levels following HR. (A) Quantitative RT-PCR analysis of p38MAPK isoform expression in HL-1 cells (n = 3). (B-D) Effect of p38MAPKα knockdown on downstream signaling and mitochondrial ROS production. 72 hours after transfection with p38MAPKα siRNAs (250 nM) or control siRNAs (250 nM), HL-1 cells were exposed to the following HR protocol: hypoxia (1 hour) and reoxygenation (15 min) and analyzed for the expression of p38MAPKα (B), phosphorylation of MK2 and ATF2 (C) and mitochondrial ROS levels (D) as described in Methods. Representative immunoblots and summary graphs are shown (B-D). The data are expressed as mean ± SEM (n = 3-4). **p < 0.01, ***p < 0.001 control siRNAs transfected cells vs. control siRNA transfected cells undergoing HR; §p < 0.01, #p < 0.001 control siRNA transfected HL-1 cells vs. p38MAPKα siRNA transfected cells, subjected to HR.