probdnf (Alomone Labs)
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probdnf
Probdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/probdnf/product/Alomone Labs
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
Probdnf, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/probdnf/product/Alomone Labs
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
probdnf - by Bioz Stars,
2022-01
93/100 stars
Images
1) Product Images from "p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex"
Article Title: p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex
Journal: The Journal of Neuroscience
doi: 10.1523/JNEUROSCI.2881-18.2019
Figure Legend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons. A , Experimental approach. B , The intensity of perisomatic PV immunostaining (green) is reduced in the binocular visual cortex ipsilateral to the minipump-releasing mut-proBDNF (Ipsi) compared with the contralateral cortex (Contra) in the same animal. On the other hand, perisomatic PV intensity in the ipsilateral cortex of PV_Cre;p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. C , Low ( C1 ) and high ( C2 ) magnification of PNN (red, WFA staining) enwrapping PV cells (green) shows a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effect is abolished in PV_Cre;p75 flx/flx mice. Scale bars: C1 , 100 μm; B , C2 , 10 μm. D , Quantification of the mean intensity of perisomatic PV-positive puncta in ipsilateral compared with contralateral cortex. I/C ratio is obtained for each animal and then averaged between different animals. Mean I/C ratio is significantly reduced in Mut-proBDNF-infused p75 Ctrl mice compared with Mut-proBDNF-infused PV_Cre;p75 flx/flx mice (unpaired t test, df = 8, t = 6.077, p = 0.0003). E , The ratio of mean PNN intensity around PV cells in ipsilateral versus contralateral cortex is significantly lower in p75 Ctrl than PV_Cre;p75 flx/flx mice infused with mut-proBDNF (unpaired t test, df = 8, t = 15.33, p
Techniques Used: Activation Assay, Immunostaining, Mouse Assay, Staining
Figure Legend Snippet: Modulation of tPA activity affects the formation of PV cell innervations during early postnatal development. A , Control EP18 PV cell ( A1 , green represents Ctrl). B , PV cell treated with the tPA inhibitor PPACK from EP10–EP18 shows simpler axonal arborization, contacting less potential targets ( B2 , blue represents NeuN-positive somata). C , PV cell treated with tPA in the same time window shows a very complex axonal arbor ( C2 ) and an increase in both terminal branching and perisomatic boutons ( C3 , arrowheads) compared with control cells ( A2 , A3 ). D , PV cell treated simultaneously with tPA and mut-proBDNF shows axonal branching and perisomatic innervation more similar to those formed by PV cell treated with mut-proBDNF alone, suggesting that the effects of tPA application may be mediated by a decrease in endogenous proBDNF/mBDNF ratio. Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–D1 , 50 μm; A2–D2 , 10 μm; A3–D3 , 5 μm. E , Perisomatic boutons density (one-way ANOVA, F (3,20) = 121.2, p
Techniques Used: Activity Assay
Figure Legend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells restores ocular dominance plasticity in adult visual cortex in vivo . A , Typical VEP responses to the stimulation of either contralateral (blue) or ipsilateral (red) eye to the cortex in which the recording is performed in p75NTR Ctrl mice infused with either vehicle or mut-proBDNF, and PV_Cre;p75NTR flx/flx mice infused with mut-proBDNF. Calibration bars: 50 μV, 100 ms. B , C/I VEP ratio mean values. Three days of monocular deprivation do not affect the C/I VEP ratio in adult mice, although it leads to a significant decrease in the C/I VEP ratio in animals treated with mut-proBDNF. Mut-proBDNF effects are, however, abolished in PV_Cre;p75 flx/flx mice (one-way ANOVA, F (2,18) = 8.903, p = 0.0021). p75NTR Ctrl + vehicle: n = 9 mice; p75NTR Ctrl + mut-proBDNF: n = 5 mice; PV_Cre;p75 flx/flx +mut-proBDNF: n = 7 mice. C , ODI of p75NTR Ctrl mice infused with vehicle solution and PV_Cre;p75 flx/flx mice infused with mut-proBDNF are not significantly different from those of undeprived animals, whereas ODIs in p75 Ctrl mice treated with mut-proBDNF are significantly shifted toward the open eye (one-way ANOVA, F (2,443) = 5.203, p = 0.0058). D , Mean spontaneous discharge is significantly increased only in p75 Ctrl mice treated with mut-proBDNF (one-way ANOVA, F (2,443) = 4.580, p = 0.0107). p75NTR Ctrl + vehicle: n = 9 mice, 174 cells; p75NTR Ctrl + mut-proBDNF: n = 5 mice, 147 cells; PV_Cre;p75 flx/flx +mut-proBDNF: n = 6 mice, 125 cells. Gray area represents the C/I VEP ratio ( B ) or the ODI range ( C ) (mean ± SEM) in adult nondeprived animals ( n = 5 mice, 99 cells). * indicate p
Techniques Used: Activation Assay, In Vivo, Mouse Assay
Figure Legend Snippet: mut-proBDNF destabilizes PV cell innervation, even after it has reached maturity. A , Control PV cell ( A1 , Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons ( A2 ), and terminal branches with prominent and clustered boutons ( A3 ; arrowheads) around NeuN-positive somata (blue). B , PV cell treated with wt-proBDNF from EP26-EP32 shows overall similar axon size ( B1 ), percentage of potentially targeted neurons ( B2 ), and perisomatic innervations ( B3 ) as control, untreated PV cells. C , PV cell treated with mut-proBDNF from EP26-EP32 shows a drastic reduction both in percentage of innervated cells ( C2 ) and perisomatic innervation ( C3 ). Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–C1 , 50 μm; A2–C2 , 10 μm; A3–C3 , 5 μm. D , Perisomatic bouton density (one-way ANOVA, F (2,18) = 93.34, p
Techniques Used:
2) Product Images from "Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator"
Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator
Journal: Scientific Reports
doi: 10.1038/srep27171
Figure Legend Snippet: Exogenous proBDNF induces pain hypersensitivity and spinal cord activation in mice. ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P
Techniques Used: Activation Assay, Mouse Assay, Injection
Figure Legend Snippet: Polyclonal Ab-proBDNF pretreatment attenuates inflammatory pain in mice. ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P
Techniques Used: Mouse Assay, Injection
Figure Legend Snippet: Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P
Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Western Blot, Migration
Figure Legend Snippet: Upregulation of p75NTR and effect of local proBDNF injection on inflammatory reaction in mice. ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p
Techniques Used: Injection, Mouse Assay, Western Blot, Expressing
Figure Legend Snippet: Upregulation of proBDNF in the local tissue in acute and persistent inflammatory pain in mice. ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p
Techniques Used: Mouse Assay, Western Blot, Injection
3) Product Images from "BDNF–TrkB signaling in the nucleus accumbens shell of mice has key role in methamphetamine withdrawal symptoms"
Article Title: BDNF–TrkB signaling in the nucleus accumbens shell of mice has key role in methamphetamine withdrawal symptoms
Journal: Translational Psychiatry
doi: 10.1038/tp.2015.157
Figure Legend Snippet: Depression-like behavior and levels of proBDNF and BDNF in the brain regions after withdrawal from repeated METH exposure. ( a ) Schedule of treatment and behavioral tests. Saline (10 ml kg −1 per day for 5 days) or METH (3 mg kg −1 per day for 5 days) was injected into mice. Behavioral tests were performed at days 7, 11 and 18 (SPT), and days 8, 12 and 19 (LMT, TST, FST). ( b ) SPT: sucrose preference of METH-treated mice was significantly lower than that of control (saline-treated) mice. LMT: there were no differences between control and METH-treated mice. TST and FST: the immobility time of METH-treated mice was significantly higher than that of control mice. * P
Techniques Used: Injection, Mouse Assay, Single-particle Tracking
4) Product Images from "The decline in synaptic GluN2B and rise in inhibitory neurotransmission determine the end of a critical period"
Article Title: The decline in synaptic GluN2B and rise in inhibitory neurotransmission determine the end of a critical period
Journal: Scientific Reports
doi: 10.1038/srep34196
Figure Legend Snippet: CS synapses in the ventral spinal cord were eliminated upon increasing synaptic 2B. ( A ) Reduction of CS synaptic activity in the ventral side after the critical period in 2a −/− mice. Spatial distribution of CS synapses determined using optical CS-EPSPs in WT, proBDNF-treated or 2a −/− mice at 11–13 or 14–16 DIV. Scale bar = 250 μm. ( B ) Ventrodorsal ratios of optical CS-EPSPs in the indicated culture pairs from 11–13 DIV (0.40 ± 0.03 in WT mice, n = 12, Ns = 12, Nm = 6; 0.42 ± 0.03 in 2a −/− mice, n = 6, Ns = 6, Nm = 6) or from 14–16 DIV (0.46 ± 0.02 in WT mice, n = 17, Ns = 17, Nm = 8; 0.39 ± 0.02 in proBDNF-treated mice, n = 4, Ns = 4, Nm = 4; 0.31 ± 0.03 in 2a −/− mice, n = 7, Ns = 7, Nm = 6).
Techniques Used: Activity Assay, Mouse Assay
Figure Legend Snippet: Decline in 2B-containing NMDAR across the end of the critical period. ( A ) Developmental alterations of the total NMDA component of CS-EPSCs in the spinal cord. Averaged CS-EPSC traces recorded after application of NBQX to WT ( upper ) or proBDNF-treated ( middle ) and 2a −/− ( lower ) mice before ( black ) or after ( red ) Ro25-6981 treatment on 12 DIV. Calibration: 50 pA, 100ms. ( B ) Averaged 2B-CS-EPSCs in WT (55.0 ± 14.6 pA, 6–8 DIV, n = 21, Ns = 16, Nm = 32; 18.0 ± 1.99 pA, 12–15 DIV, n = 18, Ns = 10, Nm = 20), proBDNF-treated (44.1 ± 6.45 pA, 12–15 DIV, n = 13, Ns = 8, Nm = 16) and 2a −/− mice (54.2 ± 10.8 pA, 6–8 DIV, n = 7, Ns = 7, Nm = 14; 74.4 ± 30.1 pA, 12–15 DIV, n = 7, Ns = 6, Nm = 12). ( C ) Developmental alterations of synaptic 2B in WT or 2a −/− mice. Immunoblot analysis of synaptic expression of 2B (2B in SPMs), PSD-95 (in SPMs) and MAGUKs (in SPMs) in WT and 2a −/− mice. Full-length blots are presented in Supplementary Fig. 7S . (D) Ratio of 2B to PSD-95 or MAGUKs in SPMs: To quantitate synaptic 2B, the intensity of 2B in SPMs was normalized to that of PSD-95 or MAGUK in SPMs in WT or 2a −/− spinal cords (Ratio of 2B to PSD-95: 1.00, 6 DIV in WT mice; 0.44, 13 DIV in WT mice; 0.62, 6 DIV in 2a −/− mice; 0.66, 13 DIV in 2a −/− mice. Ratio of 2B to MAGUKs: 1.00, 6 DIV in WT mice; 0.48, 13 DIV in WT mice; 0.75, 6 DIV in 2a −/− mice; 0.66, 13 DIV in 2a −/− mice. Each sample represents an SPM extract prepared from 20 co-cultures from 4 mice). Data are represented as the mean ± standard error of the mean. Asterisks indicate statistical significance (Student t test). * p
Techniques Used: Mouse Assay, Expressing
Figure Legend Snippet: CS axons in the ventral spinal cord regressed upon increasing synaptic 2B. ( A ) Images of CS axons labeled with ChR2-EYFP in WT ( left ), proBDNF-treated and 2a −/− spinal explants ( right ). The first image was taken at 12 DIV and the second at 15 or 16 DIV. Enumeration of CS axons on the ventral side that crossed the 70% line ( red bold line ) from the dorsal to the ventral edge of spinal gray matter. Scale bar = 250 µm. ( B ) Ratios of CS axons on the ventral side at 15 or 16 DIV to that at 12 DIV (15 DIV/12 DIV: 1.13 ± 0.06 in WT mice, n = 9, Ns = 9, Nm = 4; 16 DIV/12 DIV: 0.60 ± 0.05 in proBDNF-treated mice, n = 11, Ns = 11, Nm = 6; 0.81 ± 0.05 in 2a −/− mice, n = 11, Ns = 11, Nm = 8) ( right ).
Techniques Used: Labeling, Mouse Assay
5) Product Images from "HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology"
Article Title: HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology
Journal: PLoS ONE
doi: 10.1371/journal.pone.0150601
Figure Legend Snippet: Endocytosis of HBpF-proBDNF in Hippocampal neurons. 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 t -test, * indicates a p-value
Techniques Used: Microscopy, Staining, Marker, Fluorescence, Software, Two Tailed Test
Figure Legend Snippet: HBpF-proBDNF inhibits carbachol (CCh)-induced persistent firing in cortical pyramidal neurons. 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 t -test, * indicates a p-value
Techniques Used: Activity Assay, Produced, Two Tailed Test
Figure Legend Snippet: HBpF-proBDNF can be isolated by a modified tandem affinity purification protocol. 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.
Techniques Used: Isolation, Modification, Affinity Purification, Transfection, Expressing, Plasmid Preparation, FLAG-tag
Figure Legend Snippet: HBpF-proBDNF induces growth cone collapse. 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 t -test, * indicates a p-value
Techniques Used: Expressing, Negative Control, Two Tailed Test
Figure Legend Snippet: HBpF-proBDNF design and production. A. Schematic representation of recombinant HBpF-proBDNF protein. HBpF-proBDNF contains a signal peptide, an amino-terminal 6xHis-tag, followed by a Biotin-Acceptor-Peptide (BAP) sequence, a linker (L), a PreScission ™ Protease (PP) cleavage site and a Flag-tag (Flag). The ProBDNF sequence has been mutated with a KR to AA mutation at the furin dibasic cleavage site between the pro-domain and the mature part of BDNF. B. HEK293T cells were transfected with HBpF-proBDNF and BirA plasmids. After Ni-NTA pulldown and cleavage with the PreScission ™ Protease, the eluates were analyzed by Western blot and blotted with anti-biotin, anti-Flag and anti-BDNF. C. Purified HBpF-proBDNF was incubated with PC12 cells lysates, without a protease inhibitors, for 1h at room temperature. Incubation of HBpF-proBDNF with lysis buffer for 1h at room temperature was used as a control. Immunoblots against BDNF were performed to visualize the degradation of HBpF-proBDNF.
Techniques Used: Recombinant, Sequencing, FLAG-tag, Mutagenesis, Transfection, Western Blot, Purification, Incubation, Lysis
Figure Legend Snippet: HBpF-proBDNF interacts with endogenous p75NTR and SorCS2 in PC12 cells. 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.
Techniques Used:
Figure Legend Snippet: HBpF-proBDNF does not activate TrkB receptors. A. Following 2 days in culture, cerebellar granule neurons were stimulated with different concentrations of HBpF-proBDNF (2ng/ml; 25ng/ml and 100ng/ml) or proBDNF (2ng/ml and 25ng/ml) for 30min. After the incubation time, cells were lysed immediately in sample buffer and analyzed by Western blot against phospho-Trk and TrkB. For positive and negative controls, CGN were treated with BDNF (25ng/ml) or with BirA Ni-NTA eluate (BirA), as indicated.
Techniques Used: Incubation, Western Blot
6) Product Images from "Dexmedetomidine Attenuates Neurotoxicity in Developing Rats Induced by Sevoflurane through Upregulating BDNF-TrkB-CREB and Downregulating ProBDNF-P75NRT-RhoA Signaling Pathway"
Article Title: Dexmedetomidine Attenuates Neurotoxicity in Developing Rats Induced by Sevoflurane through Upregulating BDNF-TrkB-CREB and Downregulating ProBDNF-P75NRT-RhoA Signaling Pathway
Journal: Mediators of Inflammation
doi: 10.1155/2020/5458061
Figure Legend Snippet: Dexmedetomidine could decrease the level of proBDNF and restore the ratio of proBDNF/mBDNF and alleviates activation of the proBDNF-P75NRT-RHOA pathway after sevoflurane. (a) Western blot band. (b) ProBDNF/mBDNF. (c–e) Bar graph of Western blot. (f) Immunofluorescence of proBDNF (scale bar = 50 μ m). ∗ Compare with the control group, P
Techniques Used: Activation Assay, Western Blot, Immunofluorescence
7) Product Images from "Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF"
Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF
Journal: Frontiers in Immunology
doi: 10.3389/fimmu.2020.575607
Figure Legend Snippet: Molar concentrations of proBDNF are lower in platelets and higher in plasma than those of BDNF. ELISA quantification of proBDNF and BDNF levels in the intraplatelet (A, B) and plasma (C, D) compartments. Concentrations are normalized for 250 x 10 6 platelets. Horizontal bar represents median, *p
Techniques Used: Enzyme-linked Immunosorbent Assay
Figure Legend Snippet: Human platelets contain proBDNF. (A) ProBDNF immunoblotting of human washed platelet lysates (15 µg) from six different healthy volunteers. Recombinant proBDNF (3 ng) and human cortex lysate (3 µg) were used as positive controls. Molecular weight is indicated on the left (kDa) and primary antibody on the right. Experiments representative of n=9 for R-176 and n=10 for mab31751 antibody. IB, immunoblotting. (B) Immunoblotting of proBDNF and BDNF in different fractions of washed human platelets. P-Selectin was used as control protein in the membrane fraction, p65 NF-ĸB was used as control protein in the cytosol, and α-tubulin was used as control protein in the cytoskeleton. The equivalent of the protein content of 3 x 10 7 platelets was loaded for each fraction on the gel. Representative experiment of n=4 different volunteers. (C) ProBDNF treatment with PNGase F in washed human platelet lysates. U87-MG glioblastoma cells were used as a control. rProBDNF, recombinant proBDNF (3 ng); cortex, human cortex lysate (3 µg); platelets, whole human platelet lysate (representative experiment of n=4 different volunteers, 7.5 x 10 8 platelets per well); −PNGF, platelets treated with GlycoBuffer, and incubated at 37°C for 60 min without PNGase F; +PNGF, platelets treated with GlycoBuffer and incubated at 37°C for 60 min with PNGase F; PNGF alone, PNGase F incubated at 37°C for 60 min without platelet lysate. CD42b and sortilin were used as controls of protein deglycosylation in platelets and in U87-MG cells, respectively. n=3 different volunteers for PNGase treatments in human platelets and n=4 independent experiments for U87-MG cells. (D) Representative flow cytometry experiment showing surface and intracellular proBDNF in human washed platelets and in U87-MG and U251-MG glioblastoma cell lines. Mouse IgG 2b was used as isotype control. Percentage of expression are indicated on the figure. n=10 different healthy volunteers for human platelets; n=3 independent experiments for each glioblastoma cell line. (E) Confocal microscopy imaging of proBDNF in human permeabilized washed platelets (top) and in permeabilized U-251 MG cells (bottom). Mouse IgG 2b was used as isotype control. ProBDNF was labelled using Alexa488 fluorochrome (in green). Nuclei were stained with DAPI (in blue). Scale bar = 2 µm and 200 µm for washed platelets and U-251 MG cells images, respectively. (F) Immunoblotting of α 2 -macroglobulin at increasing quantities of loaded proteins (1–20 µg) obtained from a washed platelet lysate or platelet-poor plasma (PPP) from the same individual (#23). α-tubulin was used as loading control. Molecular weight is indicated on the left (kDa) and primary antibody on the right. PPP, platelet poor plasma; PLTs, platelets; IB, immunoblotting.
Techniques Used: Recombinant, Molecular Weight, Incubation, Flow Cytometry, Expressing, Confocal Microscopy, Imaging, Staining
Figure Legend Snippet: Unlike BDNF, intraplatelet proBDNF is not released during platelet activation. Intraplatelet (A, D) and plasma (B, E) concentrations of BDNF and proBDNF following platelet activation by different agonists. Intraplatelet concentrations are normalized for 250 x 10 6 platelets. Proportion of BDNF (C) and proBDNF (F) in plasma vs . in platelets are expressed in percentage. Error bar represents IQR, *p
Techniques Used: Activation Assay
8) Product Images from "HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology"
Article Title: HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology
Journal: PLoS ONE
doi: 10.1371/journal.pone.0150601
Figure Legend Snippet: Endocytosis of HBpF-proBDNF in Hippocampal neurons. 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 t -test, * indicates a p-value
Techniques Used: Microscopy, Staining, Marker, Fluorescence, Software, Two Tailed Test
Figure Legend Snippet: HBpF-proBDNF inhibits carbachol (CCh)-induced persistent firing in cortical pyramidal neurons. 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 t -test, * indicates a p-value
Techniques Used: Activity Assay, Produced, Two Tailed Test
Figure Legend Snippet: HBpF-proBDNF can be isolated by a modified tandem affinity purification protocol. 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.
Techniques Used: Isolation, Modification, Affinity Purification, Transfection, Expressing, Plasmid Preparation, FLAG-tag
Figure Legend Snippet: HBpF-proBDNF induces growth cone collapse. 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 t -test, * indicates a p-value
Techniques Used: Expressing, Negative Control, Two Tailed Test
Figure Legend Snippet: HBpF-proBDNF design and production. A. Schematic representation of recombinant HBpF-proBDNF protein. HBpF-proBDNF contains a signal peptide, an amino-terminal 6xHis-tag, followed by a Biotin-Acceptor-Peptide (BAP) sequence, a linker (L), a PreScission ™ Protease (PP) cleavage site and a Flag-tag (Flag). The ProBDNF sequence has been mutated with a KR to AA mutation at the furin dibasic cleavage site between the pro-domain and the mature part of BDNF. B. HEK293T cells were transfected with HBpF-proBDNF and BirA plasmids. After Ni-NTA pulldown and cleavage with the PreScission ™ Protease, the eluates were analyzed by Western blot and blotted with anti-biotin, anti-Flag and anti-BDNF. C. Purified HBpF-proBDNF was incubated with PC12 cells lysates, without a protease inhibitors, for 1h at room temperature. Incubation of HBpF-proBDNF with lysis buffer for 1h at room temperature was used as a control. Immunoblots against BDNF were performed to visualize the degradation of HBpF-proBDNF.
Techniques Used: Recombinant, Sequencing, FLAG-tag, Mutagenesis, Transfection, Western Blot, Purification, Incubation, Lysis
Figure Legend Snippet: HBpF-proBDNF interacts with endogenous p75NTR and SorCS2 in PC12 cells. 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.
Techniques Used:
Figure Legend Snippet: HBpF-proBDNF does not activate TrkB receptors. A. Following 2 days in culture, cerebellar granule neurons were stimulated with different concentrations of HBpF-proBDNF (2ng/ml; 25ng/ml and 100ng/ml) or proBDNF (2ng/ml and 25ng/ml) for 30min. After the incubation time, cells were lysed immediately in sample buffer and analyzed by Western blot against phospho-Trk and TrkB. For positive and negative controls, CGN were treated with BDNF (25ng/ml) or with BirA Ni-NTA eluate (BirA), as indicated.
Techniques Used: Incubation, Western Blot
9) Product Images from "Human Immunodeficiency Virus-1 Alters Brain-derived Neurotrophic Factor Processing in neurons"
Article Title: Human Immunodeficiency Virus-1 Alters Brain-derived Neurotrophic Factor Processing in neurons
Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience
doi: 10.1523/JNEUROSCI.0865-12.2012
Figure Legend Snippet: Neuronal localization of proBDNF in human brains
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Figure Legend Snippet: pBDNF and proBDNF levels in HIV subjects
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Figure Legend Snippet: gp120 alters the release of mBDNF/proBDNF in cortical neurons
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Figure Legend Snippet: gp120 increases proBDNF
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10) Product Images from "p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex"
Article Title: p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex
Journal: The Journal of Neuroscience
doi: 10.1523/JNEUROSCI.2881-18.2019
Figure Legend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons. A , Experimental approach. B , The intensity of perisomatic PV immunostaining (green) is reduced in the binocular visual cortex ipsilateral to the minipump-releasing mut-proBDNF (Ipsi) compared with the contralateral cortex (Contra) in the same animal. On the other hand, perisomatic PV intensity in the ipsilateral cortex of PV_Cre;p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. C , Low ( C1 ) and high ( C2 ) magnification of PNN (red, WFA staining) enwrapping PV cells (green) shows a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effect is abolished in PV_Cre;p75 flx/flx mice. Scale bars: C1 , 100 μm; B , C2 , 10 μm. D , Quantification of the mean intensity of perisomatic PV-positive puncta in ipsilateral compared with contralateral cortex. I/C ratio is obtained for each animal and then averaged between different animals. Mean I/C ratio is significantly reduced in Mut-proBDNF-infused p75 Ctrl mice compared with Mut-proBDNF-infused PV_Cre;p75 flx/flx mice (unpaired t test, df = 8, t = 6.077, p = 0.0003). E , The ratio of mean PNN intensity around PV cells in ipsilateral versus contralateral cortex is significantly lower in p75 Ctrl than PV_Cre;p75 flx/flx mice infused with mut-proBDNF (unpaired t test, df = 8, t = 15.33, p
Techniques Used: Activation Assay, Immunostaining, Mouse Assay, Staining
Figure Legend Snippet: Modulation of tPA activity affects the formation of PV cell innervations during early postnatal development. A , Control EP18 PV cell ( A1 , green represents Ctrl). B , PV cell treated with the tPA inhibitor PPACK from EP10–EP18 shows simpler axonal arborization, contacting less potential targets ( B2 , blue represents NeuN-positive somata). C , PV cell treated with tPA in the same time window shows a very complex axonal arbor ( C2 ) and an increase in both terminal branching and perisomatic boutons ( C3 , arrowheads) compared with control cells ( A2 , A3 ). D , PV cell treated simultaneously with tPA and mut-proBDNF shows axonal branching and perisomatic innervation more similar to those formed by PV cell treated with mut-proBDNF alone, suggesting that the effects of tPA application may be mediated by a decrease in endogenous proBDNF/mBDNF ratio. Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–D1 , 50 μm; A2–D2 , 10 μm; A3–D3 , 5 μm. E , Perisomatic boutons density (one-way ANOVA, F (3,20) = 121.2, p
Techniques Used: Activity Assay
Figure Legend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells restores ocular dominance plasticity in adult visual cortex in vivo . A , Typical VEP responses to the stimulation of either contralateral (blue) or ipsilateral (red) eye to the cortex in which the recording is performed in p75NTR Ctrl mice infused with either vehicle or mut-proBDNF, and PV_Cre;p75NTR flx/flx mice infused with mut-proBDNF. Calibration bars: 50 μV, 100 ms. B , C/I VEP ratio mean values. Three days of monocular deprivation do not affect the C/I VEP ratio in adult mice, although it leads to a significant decrease in the C/I VEP ratio in animals treated with mut-proBDNF. Mut-proBDNF effects are, however, abolished in PV_Cre;p75 flx/flx mice (one-way ANOVA, F (2,18) = 8.903, p = 0.0021). p75NTR Ctrl + vehicle: n = 9 mice; p75NTR Ctrl + mut-proBDNF: n = 5 mice; PV_Cre;p75 flx/flx +mut-proBDNF: n = 7 mice. C , ODI of p75NTR Ctrl mice infused with vehicle solution and PV_Cre;p75 flx/flx mice infused with mut-proBDNF are not significantly different from those of undeprived animals, whereas ODIs in p75 Ctrl mice treated with mut-proBDNF are significantly shifted toward the open eye (one-way ANOVA, F (2,443) = 5.203, p = 0.0058). D , Mean spontaneous discharge is significantly increased only in p75 Ctrl mice treated with mut-proBDNF (one-way ANOVA, F (2,443) = 4.580, p = 0.0107). p75NTR Ctrl + vehicle: n = 9 mice, 174 cells; p75NTR Ctrl + mut-proBDNF: n = 5 mice, 147 cells; PV_Cre;p75 flx/flx +mut-proBDNF: n = 6 mice, 125 cells. Gray area represents the C/I VEP ratio ( B ) or the ODI range ( C ) (mean ± SEM) in adult nondeprived animals ( n = 5 mice, 99 cells). * indicate p
Techniques Used: Activation Assay, In Vivo, Mouse Assay, Mass Spectrometry
Figure Legend Snippet: mut-proBDNF destabilizes PV cell innervation, even after it has reached maturity. A , Control PV cell ( A1 , Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons ( A2 ), and terminal branches with prominent and clustered boutons ( A3 ; arrowheads) around NeuN-positive somata (blue). B , PV cell treated with wt-proBDNF from EP26-EP32 shows overall similar axon size ( B1 ), percentage of potentially targeted neurons ( B2 ), and perisomatic innervations ( B3 ) as control, untreated PV cells. C , PV cell treated with mut-proBDNF from EP26-EP32 shows a drastic reduction both in percentage of innervated cells ( C2 ) and perisomatic innervation ( C3 ). Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–C1 , 50 μm; A2–C2 , 10 μm; A3–C3 , 5 μm. D , Perisomatic bouton density (one-way ANOVA, F (2,18) = 93.34, p
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