METH decreased basal pERK1/2 levels and potentiated ERK1/2 phosphorylation induced by 6-OHDA.

<p>(A) Western blot analysis showing pERK levels at different time points after exposure to sub-lethal concentration of METH (0.5 mM) for up to 24 hr. Lysates were collected at 15 min, 30 min, 1 hr, 6 hr, and 24 hr. Shown are the 15 min and 30 min time points. (B) pERK1/2 levels decreased by 15 min post METH exposure and lasted for up to 24 hr. METH decreased activation of ERK 1/2 in a concentration-dependent manner (0, 0.5, and 1 mM at the 24 hr time point) and enhanced the activation of ERK1/2 after 6-OHDA exposure. This activation lasted from 15 to at least 24 hr. (C) Quantification of ERK1/2 activation in response to 6-OHDA. (D) MN9D cells were treated with sub-lethal concentration of METH (0.5 mM) for 24 hr, the medium was removed and the cells treated with the MEK1/2 inhibitor U0126 (10 µM) for 1 hr before and during the 20 min 6-OHDA exposure. Western blot analysis confirmed the activation of ERK1/2 by 6-OHDA and its enhancement by METH preconditioning and confirmed that U0126 blocked the phosphorylation of ERK1/2. (E) Viability assay looking at ATP levels and performed 24 hr post 6-OHDA treatment showed that METH protected MN9D cells against 6-OHDA toxicity and U0126 abolishes this protection. α-tubulin was used as a loading control. Data are presented as means ± SEM (N = 3). *p<0.05, **p<0.01 compared to vehicle and ^#p<0.05 compared to lane 10.