Figure 3
Recombinant CR-proBDNF induces apoptosis of cerebellar granule neurons cultured in low K+-containing medium. (A) Preparation of recombinant CR-proBDNF (R125M/R127L-BDNF: proBDNFML) using the Baculovirus or E. coli expression systems. Protein products were analyzed by silver staining (left) and Western blotting using rabbit antibody against BDNF prodomain. (B-C) Opposite actions of CR-proBDNF and matBDNF revealed by apoptosis assay. After 4 days of culture in HK medium, CGNs were treated with E. coli-derived CR-proBDNF or matBDNF in LK medium. Cell death was assessed using DAPI staining 48 h after treatment with the indicated drug in LK medium. (B) Arrows and arrowheads indicate representative living and dead cells, respectively. Scale bar, 50 μm. (C) For quantification of cell death, 300–400 cells were counted in four independent fields per coverslip. (D) Dose-dependent test of pro-apoptotic activity of CR-proBDNF. Cell death was assessed using DAPI staining 48 h after treatment with CR-proBDNF in LK medium. DAPI data were expressed as the percentage of mock cultures. In the cell death assay (C-D), n = 4 independent culture dishes. ANOVA followed by post-hoc analysis, *P < 0.05; **P < 0.01. Results were replicated in at least three independent experiments.