Figure 3. The stimulatory effect of caloric restriction, NPY, and ghrelin on autophagy in cortical neurons is mediated by the activation of ERK1/2-MAPK. Primary rat cortical neurons were exposed to caloric restriction mimic medium (CR) - DMEM low glucose, NPY (100 nM), or ghrelin (GHRL, 10 nM) for 6 h. Untreated cells were used as control (Ctrl). (A–I) Cells were incubated with ERK1/2-MAPK inhibitor (U0126 (U0), 1 μM), 30 min before caloric restriction, NPY or ghrelin treatment. Whole-cell extracts were assayed, phospho-ERK (A, D, G), LC3B-II (B, E, H), SQSTM1 (C, F, I), and ERK or α-Actin (loading control) immunoreactivity through Western blotting analysis, as described in Materials and Methods. Representative Western blots for each protein are presented above each respective graph. The results represent the mean±SEM of, at least, four independents experiments, and are expressed as a percentage of control. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001, significantly different compared to control; $p<0.05, $$p<0.01, $$$p<0.001 and $$$$p<0.0001, significantly different from stimulus-treated cells, as determined by ANOVA, followed by Newman-Keuls multiple comparison test.