FIGURE 6.

TrkB receptor internalization in primary neurons. A and B, TrkB receptor internalization and sorting to early endosomes. Primary cultured cortical neurons were incubated with anti-TrkB antibody and FITC-conjugated secondary antibody (3 μg/ml). The cells were incubated with 20 nm BDNF or 2.5 μm 7,8-DHF for 10 or 60 min at 37 °C. The cells were then fixed in 4% paraformaldehyde and stained with mouse anti-EEA1 antibody and Alexa Fluor 555-conjugated goat anti-mouse secondary antibody. Scale bar = 10 μm. Quantitative analysis of internalized TrkB receptors was performed with results from independent experiments. *, p < 0.05; **, p < 0.01; Student's t test; n = 3. C, the effect of BDNF and 7,8-DHF on the internalization of TrkB as determined by surface biotinylation. Cell surface proteins were labeled by Sulfo-NHS-SS-Biotin before initiation of TrkB internalization by BDNF or 7,8-DHF for 30 min at 37 °C. The remaining biotinylated proteins on the cell surface were removed by glutathione. The internalized biotin-modified TrkB receptors were precipitated by streptavidin, followed by Western blot analysis using TrkB antibody. 7,8-DHF and BDNF enhanced TrkB internalization (full-length (145-kDa) and truncated (95-kDa) TrkB receptors) in primary neurons. The quantitative data were mean ± S.D. from two independent experiments. *, p < 0.05; ***, p < 0.001; Student's t test. M.W., molecular weight; IP, immunoprecipitation; IB, immunoblot.