recombinant human probdnf  (Alomone Labs)

Journal: The Journal of Neuroscience

Article Title: SPIG1 Negatively Regulates BDNF Maturation

doi: 10.1523/JNEUROSCI.1597-13.2014

Figure Lengend Snippet: Effect of SPIG1 on BDNF expression in PC12 cells. A , Colocalization of SPIG1 and proBDNF in vesicle-like structures. Twelve hours after transfection with the indicated constructs, PC12 cells were differentiated with NGF as described in Materials and Methods.

Article Snippet: A 96-well polystyrene ELISA plate (#9018, Costar) was coated with 4 pmol of purified recombinant human proBDNF (B-257, Alomone Labs) or mature BDNF (GF029, Millipore).

Techniques: Expressing, Transfection, Construct

Journal: The Journal of Neuroscience

Article Title: SPIG1 Negatively Regulates BDNF Maturation

doi: 10.1523/JNEUROSCI.1597-13.2014

Figure Lengend Snippet: A mechanism model for the position-specific branching of RGC axons. Both ephrin-A/EphA signaling (light yellow) in the distal part and proBDNF/ephrin-A-p75 NTR signaling (yellow) in the proximal part have been shown to mediate the suppression of branching

Article Snippet: A 96-well polystyrene ELISA plate (#9018, Costar) was coated with 4 pmol of purified recombinant human proBDNF (B-257, Alomone Labs) or mature BDNF (GF029, Millipore).

Techniques:

Journal: The Journal of Neuroscience

Article Title: SPIG1 Negatively Regulates BDNF Maturation

doi: 10.1523/JNEUROSCI.1597-13.2014

Figure Lengend Snippet: Subcellular colocalization of exogenous SPIG1 and proBDNF in the chick RGC. A , Colocalization of SPIG1 and proBDNF in axon terminals. After the electroporation of SPIG1 (FLAG-tagged at the C terminus) and BDNF constructs at HH stage 9, retinal cells were

Article Snippet: A 96-well polystyrene ELISA plate (#9018, Costar) was coated with 4 pmol of purified recombinant human proBDNF (B-257, Alomone Labs) or mature BDNF (GF029, Millipore).

Techniques: Electroporation, Construct

Journal: Neural Development

Article Title: Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance

doi: 10.1186/1749-8104-5-30

Figure Lengend Snippet: Proneurotrophins abolish retinal axon branching via p75 NTR . The outgrowth/branching assay was performed as described [ 6 ]. Cells from E8 nasal retina were electroporated with eGFP and plated on a merosin/laminin substrate. (A) Cultures were treated at 1 day in vitro with 5 ng/ml BDNF, or 5 ng/ml proneurotrophins in the presence of 5 ng/ml BDNF as indicated, and fixed and analysed for branch number per axon after 3 days in vitro . The basal level of branching was not affected by treatment of retinal cultures with proneurotrophins alone. However, both proBDNF and proNGF led to a downregulation of the BDNF-induced branching to basal levels. (B) The length of outgrowth of retinal axons is not affected by treatment with neurotrophins and/or proneurotrophins. Axon length is given in arbitrary units. (C) To show that the proBDNF effect is mediated via p75 NTR , retinal cultures were electroporated either with an RNAi vector resulting in the knockdown of p75 NTR , with an RNAi vector not affecting p75 NTR protein levels or with empty vector. After plating, the cultures were treated with pro/neurotrophins as described in (A). p75 NTR knockdown obliterates the branch-suppressing effect of proBDNF. Three independent experiments were performed. The statistical analysis was done using Kruskal-Wallis test and Dunn's multiple comparison test post hoc . *** P

Article Snippet: Experimental reagents EphA7-Fc was from R & D Systems, Fc control from Calbiochem, BDNF from Promega, wild-type proBDNF and proNGF from Alomone Lab (Jerusalem, Israel).

Techniques: In Vitro, Plasmid Preparation

Journal: Neural Development

Article Title: Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance

doi: 10.1186/1749-8104-5-30

Figure Lengend Snippet: Expression of proBDNF in chick RGC axons . Retinal single cell cultures derived from E6 chick retina were stained after 2 days in vitro using a monoclonal antibody against the pro-domain of proBDNF [ 14 ] according to protocols given in Yang et al . [ 14 ]. (A-C) An RGC growth cone stained with control antibody (B), and phalloidin (C) to show the location of actin. (A) The composite of (B) and (C). In all composites phalloidin is shown in green and proBDNF/control antibody in red. (D-F) An RGC axon stained with control antibody (E), and phalloidin (F). (D) The composite of (E) and (F). (G-I) An RGC axon stained with a proBDNF antibody (H) [ 14 ], and phalloidin (I). (G) The composite of (H) and (I). (J-L) . An RGC growth cone stained with proBDNF antibody (K), and phalloidin (L). (J) The composite of (K) and (L).

Article Snippet: Experimental reagents EphA7-Fc was from R & D Systems, Fc control from Calbiochem, BDNF from Promega, wild-type proBDNF and proNGF from Alomone Lab (Jerusalem, Israel).

Techniques: Expressing, Derivative Assay, Staining, In Vitro

Journal: Neural Development

Article Title: Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance

doi: 10.1186/1749-8104-5-30

Figure Lengend Snippet: Repellent axon guidance is disrupted in the presence of an anti-proBDNF antibody or a soluble extracellular domain of p75 NTR . (A) Quantification of axon growth preferences in the presence of an anti-proBDNF antibody [ 14 ] or a control antibody. Stripe assay experiments were performed in the presence of a proBDNF antibody (1:200) [ 14 ] or a control antibody (mouse monoclonal antibody for placental alkaline phosphatase; 1:200). The quantification of axon growth preferences shows an abolishment of repellent guidance in the presence of the proBDNF antibody (see also Additional file 4 ). Error bars represent the standard error of the mean. Statistics were performed using Kruskal-Wallis test and Dunn's multiple comparison test with *** P

Article Snippet: Experimental reagents EphA7-Fc was from R & D Systems, Fc control from Calbiochem, BDNF from Promega, wild-type proBDNF and proNGF from Alomone Lab (Jerusalem, Israel).

Techniques: Stripping Membranes

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend 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

Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

Techniques: Activation Assay, Mouse Assay, Injection

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend 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

Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

Techniques: Mouse Assay, Injection

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend 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

Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay, Western Blot, Migration

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend 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

Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

Techniques: Injection, Mouse Assay, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

doi: 10.1038/srep27171

Figure Lengend 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

Article Snippet: Other recombinant human proBDNF, mice proBDNF and human mBDNF for ELISA, Western Blot or behavior studies were purchased from Alomone Labs (Israel).

Techniques: Mouse Assay, Western Blot, Injection

Journal: bioRxiv

Article Title: Prelimbic proBDNF facilitates memory destabilization by regulation of neuronal function in juveniles

doi: 10.1101/2021.12.30.474526

Figure Lengend Snippet: The increase proBDNF alters synaptic currents, promotes LFS-induced synaptic depression and strengthens the theta phase-gamma amplitude coupling during the PR-LTM test. (A) Schematic describing the timeline for morphological analysis (Top-left). Illustration of the region of interest in prelimbic images (Top-middle), and dendritic segment analysis for spine quantification (Top-right). Red circles indicated the mushroom type spine, yellow circles indicated thin type spine and blue circles indicated stubby type spine. Sample images were projected at minimal intensity and inverted, background was then subtracted, followed by brightness/contrast adjustment. Scale bars, 5 μm. Quantification of spine density (Bottom-left) and the proportion of spine (Bottom-right). No statistical difference in spine density was found between juvenile and adult groups. However, a significant higher proportion of thin type spine but a lower mushroom type spine was observed in juveniles compared with adults. (B) Schematic describing the timeline for EPSCs recordings (Top-left). Representative continuous traces (Top-middle) and average waveform (Top-right) of the pharmacologically isolated NMDA EPSCs in the prelimbic neurons of adult, juvenile and juvenile+anti groups. No change in the amplitude of EPSCs (Bottom-left) was found but the frequency (Bottom-middle) and decay time (Bottom-right) were significantly increased in juvenile group. The enhanced frequency and decay time of NMDA currents in juvenile group were inhibited after infusions of anti-proBDNF antibody. (* P

Article Snippet: Needles were inserted into bilateral cannulae and then cleavage-resistant proBDNF (2 ng/ml; Cat#B257 Alomone Labs), anti-proBDNF antibody (10 μg/μL; Cat#ANT-006, Alomone Labs), TAT-Pep5 (4 ng/μL; Cat#506181, EMD Millipore), K252a (25 μg/μL; Cat#82497; Sigma-Aldrich), 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP; 32 ng/μL; Cat#01773, Tocris Bioscience), NVP-AAM077 (0.8 ng/μL; Cat#P1999, Sigma-Aldrich), Ro25-6981 (2.0 ng/μL; Cat#1594, Tocris Bioscience), mature BDNF (1.5 μg/mL; Cat#B250; Alomone Labs) or artificial CSF (ACSF, Cat#3525, Tocris Bioscience) into prelimbic area (at a rate of 0.5 μL/min/side for 2 min) was infused immediately or one day following memory retrieval.

Techniques: Isolation

Journal: bioRxiv

Article Title: Prelimbic proBDNF facilitates memory destabilization by regulation of neuronal function in juveniles

doi: 10.1101/2021.12.30.474526

Figure Lengend Snippet: The higher prelimbic proBDNF expression during the juvenile period facilitates retrieval-dependent memory destabilization. Quantification of the proBDNF (A) and its receptor p75 NTR (B) levels in prelimbic cortex of juvenile and adult rats. Representative immunoblots the expression of proBDNF and p75 NTR (Top). A significant increase in the proBDNF levels was detected in juvenile group, as well the p75 NTR levels (Bottom). (* P

Article Snippet: Needles were inserted into bilateral cannulae and then cleavage-resistant proBDNF (2 ng/ml; Cat#B257 Alomone Labs), anti-proBDNF antibody (10 μg/μL; Cat#ANT-006, Alomone Labs), TAT-Pep5 (4 ng/μL; Cat#506181, EMD Millipore), K252a (25 μg/μL; Cat#82497; Sigma-Aldrich), 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP; 32 ng/μL; Cat#01773, Tocris Bioscience), NVP-AAM077 (0.8 ng/μL; Cat#P1999, Sigma-Aldrich), Ro25-6981 (2.0 ng/μL; Cat#1594, Tocris Bioscience), mature BDNF (1.5 μg/mL; Cat#B250; Alomone Labs) or artificial CSF (ACSF, Cat#3525, Tocris Bioscience) into prelimbic area (at a rate of 0.5 μL/min/side for 2 min) was infused immediately or one day following memory retrieval.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Prelimbic proBDNF facilitates memory destabilization by regulation of neuronal function in juveniles

doi: 10.1101/2021.12.30.474526

Figure Lengend Snippet: Up-regulation of proBDNF-p75NTR signaling mediated by NMDA-GluN2B contributes to enhance the modulation of existing fear memory traces in juvenile rats. (A) Schematic describing the behavioral timeline for the retrieval-dependent memory destabilization experiment using rats conditioned with four tones (Top). Immediately following the memory retrieval, the rats infused with TAT-Pep5, K252a or vehicle into the prelimbic cortex 15 min prior to the mBDNF, proBDNF or vehicle infusion. Two days later, PR-LTM was assessed by exposed the rats to the novel context. Similar, no significant difference in the percentage freezing during the memory retrieval but the percentage freezing level during the PR-LTM test was significant lower in juvenile group than adult group (Bottom). No obvious effect of mBDNF on freeze behavior was found. Infusions of p75 NTR blocker TAT-Pep5 could significantly enhance the percentage of freeze behavior. Meanwhile, infusions of TAT-Pep5, but not K252a, markedly blocked the effects of proBDNF treatment. (* P

Article Snippet: Needles were inserted into bilateral cannulae and then cleavage-resistant proBDNF (2 ng/ml; Cat#B257 Alomone Labs), anti-proBDNF antibody (10 μg/μL; Cat#ANT-006, Alomone Labs), TAT-Pep5 (4 ng/μL; Cat#506181, EMD Millipore), K252a (25 μg/μL; Cat#82497; Sigma-Aldrich), 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP; 32 ng/μL; Cat#01773, Tocris Bioscience), NVP-AAM077 (0.8 ng/μL; Cat#P1999, Sigma-Aldrich), Ro25-6981 (2.0 ng/μL; Cat#1594, Tocris Bioscience), mature BDNF (1.5 μg/mL; Cat#B250; Alomone Labs) or artificial CSF (ACSF, Cat#3525, Tocris Bioscience) into prelimbic area (at a rate of 0.5 μL/min/side for 2 min) was infused immediately or one day following memory retrieval.

Techniques: