Endocytosis of HBpF-proBDNF in Hippocampal neurons.

<p>Primary hippocampal neurons cells were exposed to HBpF-proBDNF (250 ng/ml) conjugated to Streptavidin-Cy3 for 1h to 6h. After fixation and mounting, cells were analyzed by fluorescent microscopy. Hoechst staining was used as a nuclear marker. Quantification of fluorescence intensity was performed using ImageJ software on three independent experiments. 100 cells were measured for each experiment. (unpaired two-tailed <i>t</i>-test, * indicates a p-value < 0.05; bars indicate standard error).</p

HBpF-proBDNF interacts with endogenous p75NTR and SorCS2 in PC12 cells.

<p>PC12 cells were stimulated with HBpF-proBDNF (100μg/ml) with or without 9μM GM6001 for 3h. BirA Ni-NTA eluate was used as control treatment. After stimulation, cells were lysed and HBpF-proBDNF and associated protein were recovered on SA beads. Cleavage with PP was performed for 16 hours and the resulting PP eluate was collected. Cell lysates (Input) and PP eluate samples were then analyzed by immunoblotting for p75NTR, SorCS2 and FLAG.</p

HBpF-proBDNF induces growth cone collapse.

<p>Following 2 days in culture, hippocampal neuronal culture were stimulated with different concentrations of HBpF-proBDNF or proBDNF (25ng/ml and 100ng/ml) for 1h. Ni-NTA eluate from cells expressing only BirA was used as a negative control. Cells were then fixed and immunostained against beta-III-tubulin (Tuj-1) and phalloidin (scale bar = 10μm). Quantification of growth cone-collapse was done on three independent experiments and 50 growth-cones were counted for each experiment (unpaired two-tailed <i>t</i>-test, * indicates a p-value < 0.05; bars indicate standard error).</p

HBpF-proBDNF inhibits carbachol (CCh)-induced persistent firing in cortical pyramidal neurons.

<p>A. Representative trace of current-clamp recording from pyramidal neuron in layer V of the entorhinal cortex. Slices were perfused with 10μM CCh and the persistent activity was produced by a short depolarization (1s, 100pA). HBpF-proBDNF at 2ng/ml was next added in presence of 10μM CCh during 10 minutes (first cut in the trace) and cells were stimulated. HBpF-proBDNF was removed by perfusing a solution containing only 10μM CCh for 10 minutes (second cut in the trace) and before the stimulation of the cells. B. Quantification of the plateau amplitude and frequency of the persistent activity (unpaired two-tailed <i>t</i>-test, * indicates a p-value < 0.05; bars indicate standard error).</p

HBpF-proBDNF can be isolated by a modified tandem affinity purification protocol.

<p>A. HEK293T cells were transfected with p75NTR, Sortilin, HBpF-proBDNF, and BirA expression plasmid as indicated. 48h after transfection, HEK293T cells were lysed (input) and pulled-down on Ni-NTA beads. The Ni-NTA eluate was then pulled-down on SA beads and then cleaved by PP overnight (PP eluate). Samples were analyzed by immunoblotting for p75NTR, sortilin, biotin and the Flag tag.</p