Figure 4
CC1-EGFP overexpression disrupted TrkB retrograde signaling. (a) Cultured hippocampal neurons (DIV7) were infected with GFP or CC1-EGFP contained lentivirus. 48 h later, neurons were starved for 8 h with serum-free medium and then were treated with BDNF (50 ng/ml) for 30 min. Cell lysates were collected and the level of Erk5 phosphorylation was detected by western blot. (b) Quantitative analysis of p-Erk5 level in (a). Data are shown as the mean±S.E.M. (n=3–4 per group, *P<0.05, compared with the EGFP group). (c and e) Hippocampal neurons infected with EGFP or CC1-EGFP constructs were cultured in the microfluidic chambers for 6 days. BDNF (50 ng/ml) or control vehicle was applied to the cell body compartment (c) or the axonal compartment (e) for 30 min. Then, cells were stained with anti-GFP (green) and anti-pErk5 (red) antibodies. Images of cell bodies are shown. Asterisk indicates EGFP or CC1-EGFP-positive neurons, and arrow indicates the uninfected wild-type neurons. Scale bar, 10 μm. (d and f) Quantitative analysis of the intensity of p-Erk5 in the cell bodies after BDNF treatment to the cell body compartment (d) or axonal compartment (f). The numbers of neurons analyzed for each group was >30. Values are shown as the mean±S.E.M. (n=3, *P<0.05, **P<0.01 compared to the vehicle-treated EGFP group; ##P<0.01 compared to the BDNF-treated EGFP group)
