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Figure 2 | Molecular Brain

Figure 2

From: Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival

Figure 2

Intracellular proteolytic cleavage of proBDNF is not a crucial step for intracellular transportation and secretion of BDNF in hippocampal neurons. (A) Distribution of GFP-tagged wild-type BDNF (BDNF-GFP) and cleavage-resistant (CR)-proBDNF (R125M/R127L-BDNF-GFP) in the cell body and processes. Cultured neurons were subjected to Sindbis viruses expressing the indicated constructs for 3 h. Three days later, the neurons were fixed for imaging of GFP fluorescence using confocal microscope. Representative low-magnification (upper) and high-magnification images of the cell body (middle) and neuronal processes (bottom) are shown. (B-C) The infected cells were immunostained with the indicated antibodies for confocal imaging. Note that CR-proBDNF-GFP greatly co-localized with TGN38 (B) and SgII (C), similarly to wild-type BDNF-GFP (arrows). Scale bar in all images, 10 μm. (D) Expression of wild-type proBDNF-Myc or CR-proBDNF-Myc. Hippocampal neurons were introduced with constructs encoding GFP and wild-type BDNF-Myc or CR-proBDNF-Myc using Sindbis virus expression system. Three-days after a brief (3 h) infection, cells were double-stained using anti-Myc and anti-proBDNF antibodies. BDNF-Myc signals largely co-localized with proBDNF signals (arrows) indicating that proBDNF is the predominant intracellular isoform. Scale bar, 5 μm.

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