FIGURE 4.

Effect of chronic BDNF treatment on the levels and phosphorylation status of eEF2. Conventional and quantitative RT-PCR was performed to examine eEF2 mRNA levels in neurons following BDNF treatment for 5 days (A). A single band of predicted size (236 bp) was observed (n = 4 in each group). Quantitative RT-PCR indicates there is no difference between control and BDNF-treated neurons (n = 4, p = 0.931 (t test)). Neurons were pulse labeled with [³⁵S]methionine for 1 h, and then washed. eEF2 was immunoprecipitated after 1 and 6 h and analyzed in neurons at 4 days in culture and quantified by Image analyzer (BAS 5000, Fuji Film). Bars represent mean ± S.D. (n = 3). *, p < 0.01 (t test) (B). Western blotting (WB) (D and E) and densitometric analysis (D and E) were performed to examine the total and phosphorylated (inactive) protein levels of eEF2 in neurons following BDNF treatment (50 ng/ml) for 5 days. Bars represent the mean ± S.D. (n = 8). *, p < 0.001 (t test). The phosphorylation ratio of eEF2 is presented in panel F.