Figure 5.

p66Shc is required for PEITC‐induced ROS production. NIH 3T3 wt and NIH 3T3 V600E cells were transfected with plasmids expressing either scrambled or p66Shc‐specific shRNA and selected with puromycin. For immunoblots, cells were seeded in six‐well plates in complete growth medium at a density of 600 000 cells per well and treated with 20 μm PEITC for 30 min prior to lysis. p66Shc expression was analyzed by immunoblotting (A) and normalized to loading control (B). Fluorescence microscopy of ROS levels was performed after 30 min of 5 μm PEITC stress and MitoTracker Red CM‐H2XRos staining. Data are presented as mean value ± standard error of the mean (SEM) of 100 measurements per sample and three experiments (C). *P < 0.1, ***P < 0.001.