Fig 4. Double phosphorylation of ERK by MEK relies almost completely on mitochondrial phospho-Thr183.

LP07 cells were transiently transfected with wild type ERK2-V5 (WT), Y183A or T183A ERK mutants, using Lipofectamine 2000 reagent. Forty-eight hours post-transfection LP07 were stimulated with 1 μM H₂O₂ during 0–30 min and then incubated in the presence or absence of 1 μM H₂O₂ for the indicated times. Then, the cytosolic, mitochondrial and nuclear fractions were obtained and the detection of ERK2 was analyzed by western blot using anti-V5 antibody. Representative images of cytosolic and mitochondrial fractions ERK2-WT (Panel A) and nuclear fraction ERK2-WT (panel D) distribution and representative images of Y185A and T185A distribution in the cytosolic (Panel B), mitochondrial (Panel C) and nuclear (Panel D) fractions, from three independent experiments. Relative levels of ERK mutants in cytosol and mitochondria relative to basal conditions, arbitrarily defined as 1 (Panels B and C). Data are expressed as the mean ± SD of three independent experiments * p<0.05, **p<0.01 vs. 0 min with H₂O₂.