probdnf protein  (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: Frontiers in Aging Neuroscience

Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

doi: 10.3389/fnagi.2017.00284

Figure Lengend Snippet: Memory impairment in aged mice positively correlates with increases in hippocampal proBDNF levels. (A) Average (±SEM) reference memory (RM) and working memory (WM) errors over four 4-session blocks of a WRAM task in 24-month old aged mice and 4-month old young mice. RM and WM errors positively correlate with proBDNF levels. (B) Increase in proBDNF, but not brain-derived neurotrophic factor (BDNF), in 24-month old aged mice relative to young 4-month old mice. (C) Relative to young 4-month old mice, aged 24-month old mice show increased p75 Neurotrophin Receptor (p75NTR) and decreased p-trk140 and trk140. (D) Relative to young 4-month old mice, aged 24-month old mice show decreased Tissue Plasminogen Activator (tPA), but not carboxypeptidase E (CPE). * p

Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

Techniques: Mouse Assay, Derivative Assay

Journal: Frontiers in Aging Neuroscience

Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

doi: 10.3389/fnagi.2017.00284

Figure Lengend Snippet: Theoretical model of the role of proBDNF in learning in memory in aged individuals. In young individuals, maturation of proBDNF to BDNF is controlled by plasmin and tPA, and is essential for learning and memory. Aged individuals show decreased levels of tPA and plasmin, and increased levels of uncleaved proBDNF, associated with increased spine remodeling and memory deficits. Blockade of p75NTR (e.g., by TAT-Pep5, as in the current study) leads to spine growth, and rescues learning and memory.

Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

Techniques:

Journal: Frontiers in Aging Neuroscience

Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

doi: 10.3389/fnagi.2017.00284

Figure Lengend Snippet: Timeline of Experiments 1 and 2. In Experiment 2, mice were split into three groups based on their performance (unimpaired/impaired) and henceforth infused with different drugs (saline, proBDNF, TAT-Pep5). d, day of study; WRAM, behavioral training/testing in the water radial arm maze task; SAL, saline.

Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

Techniques: Mouse Assay

Journal: Frontiers in Aging Neuroscience

Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

doi: 10.3389/fnagi.2017.00284

Figure Lengend Snippet: Intra-hippocampal infusion of proBDNF impairs memory in well-performing (unimpaired) mice, while TAT-Pep5 infusion improves memory in poorly-performing (impaired) mice. (A) Average (±SEM) RM and WM errors in memory impaired 18-month old mice receiving intra-hippocampal saline infusions (impaired + SAL, n = 8, open triangles) and well-performing 18-month old aged mice receiving uncleavable proBDNF intra-hippocampal infusions (unimpaired + proBDNF, n = 16, open circles) over four daily sessions of a WRAM task. (B) Average (± SEM) RM and WM errors in memory impaired 18-month old mice infused with saline (impaired + SAL, n = 8, open triangles) and memory impaired 18-month old mice receiving intra-hippocampal infusions of TAT-Pep5 (impaired + TAT-Pep5, n = 9, closed circles) over four daily sessions of a WRAM task. (C) Representative images indicating the locations of drug infusions at two levels of the hippocampus. ns not significant; * p

Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

Techniques: Mouse Assay

Journal: Frontiers in Aging Neuroscience

Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

doi: 10.3389/fnagi.2017.00284

Figure Lengend Snippet: Intra-hippocampal infusion of proBDNF increases p-cofilin levels in memory-unimpaired mice to levels seen in memory-impaired mice. (A) p-Cofilin to total cofilin ratio in memory-unimpaired mice, memory-impaired mice, and memory-unimpaired mice infused with proBDNF. (B) Representative p-cofilin and cofilin blots. ** p

Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

Techniques: Mouse Assay

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: Journal of Neuroinflammation

Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

doi: 10.1186/s12974-020-01850-0

Figure Lengend Snippet: Schematic diagram showing how proBDNF dampens CD4 + T cell activity and contributes to the pathogenesis of SAE. In sepsis, proBDNF expression is increased in peripheral blood and meningeal immune cells, which then decreases the infiltration of CD4 + T cells in the meninges. As a result, meningeal pro-inflammatory cytokines such as IL-6 and IL-1β are upregulated, but anti-inflammatory cytokines including IL-4 and IL-13 are downregulated, finally leading to SAE. SAE, sepsis-associated encephalopathy

Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

Techniques: Activity Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

doi: 10.1186/s12974-020-01850-0

Figure Lengend Snippet: Exogenous proBDNF protein reduced CD4 + T cells but increased CD8 + T cells of septic mice in vitro. Mice injected with saline or LPS (5 mg kg −1 ) for 5 days and the splenocytes were isolated and cultured for 3 days in vitro. Exogenous proBDNF did not alter the percentage of a CD3 + T cells in CD45 + cells or the percentage of b CD4 + T cells or c CD8 + T cells in CD3 + T cells in splenocytes from mice treated with saline. d Exogenous proBDNF did not alter the percentage of CD3 + T cells in CD45 + cells in splenocytes in septic mice. e–f ProBDNF treatment significantly decreased the percentage of e CD4 + T cells but increased the percentage of f CD8 + T cells in CD3 + T cells in splenocytes in LPS-treated mice. n = 4 in each group. Data were analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

Techniques: Mouse Assay, In Vitro, Injection, Isolation, Cell Culture

Journal: Journal of Neuroinflammation

Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

doi: 10.1186/s12974-020-01850-0

Figure Lengend Snippet: Increased proBDNF expression in meningeal and peripheral blood immune cells in septic mice. Mice were i.p. injected with LPS (5 mg kg −1 ) and meninges and peripheral blood were harvested for immunofluorescence staining or flow cytometry. a Representative whole mount meningeal immunofluorescence images showed markedly increased proBDNF-positive staining cells in the meninges in mice at 1 day after LPS injection compared to saline injected mice. The high magnification images around the arrows are displayed in a white square as insets. Bar = 100 μm. b – f Representative meningeal single cell flow cytometry images ( upper panel ) and its statistical analysis ( lower panel ) indicated that proBDNF MFI was increased in meningeal b CD3 + T cells, c CD4 + T cells, d CD8 + T cells, and f CD11b + monocytes/macrophages at 1 day after LPS injection. proBDNF in meningeal e CD19 + B cells upregulated until 5 days after LPS injection. n = 10 in the Con group, n = 6 in LPS groups. g – k Upregulation of proBDNF in g CD3 + T cells, h CD4 + T cells, i CD8 + T cells, j CD19 + B cells, and k CD11b + monocytes/macrophages in peripheral blood in LPS-injected mice were detected. n = 9 in Con group, n = 8 in the LPS1d group, n = 4 in the LPS 5d group. Data b – k were analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

Techniques: Expressing, Mouse Assay, Injection, Immunofluorescence, Staining, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

doi: 10.1186/s12974-020-01850-0

Figure Lengend Snippet: Effect of i.c.v. injection of anti-proBDNF antibody on fear memory and meningeal immune activity in the septic mice. Mice were bilateral i.c.v. injected with 1 μg McAb-proB 3 days before LPS injection. Behavior tests were performed 1 day after LPS injection. a Representative images showed the broad and thorough diffusion of drugs in cerebroventricular lumen following i.c.v. injection with methylene blue. b McAb-proB i.c.v. injection did not influence weight of mice. c–e There was no difference of c fear conditioning acquiring performance in each group, nor McAb-proB increased the freezing time of d contextual or e cued fear conditioning test as compared to IgG control after LPS injection. n = 6 in each group. Data b , c , and e were analyzed by repeated measures ANOVA and followed by Bonferroni post hoc test and data d was analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

Techniques: Injection, Activity Assay, Mouse Assay, Diffusion-based Assay

Journal: Journal of Neuroinflammation

Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

doi: 10.1186/s12974-020-01850-0

Figure Lengend Snippet: Systemic blockade of proBDNF ameliorated cognitive dysfunction and restored meningeal and peripheral CD4 + T cell ratio in septic mice. Mice were i.p. injected with proBDNF 30 min before LPS (5 mg kg −1 ) injection. Fear conditioning testing was performed 1 day after LPS injection. Meninges and peripheral blood were harvested 5 days after LPS injection for flow cytometry. a McAb-proB did not influence the weight of mice or b fear conditioning acquiring. c , d McAb-proB greatly alleviated memory deficit induced by LPS injection in mice as indicated by the increased freezing time in ( c ) contextual and ( d ) cued fear conditioning tests in the McAb-proB group relative to the IgG control. n = 8 in each group. Data a , b , and d were analyzed by repeated measures ANOVA and followed by Bonferroni post hoc tests. Data c was analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

Techniques: Mouse Assay, Injection, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

doi: 10.1186/s12974-020-01850-0

Figure Lengend Snippet: Systemic blockade of proBDNF restored meningeal pro-inflammatory microenvironment in septic mice. Mice were i.p. injected with proBDNF 30 min before LPS (5 mg kg −1 ) injection. Meninges were harvested 5 days after LPS injection for qPCR. a The level of CD4 gene expression was higher in the meninges of the McAb-proB group than in IgG controls in septic mice. b–f Gene levels were significantly lower in b IL-1β and c IL-6 but higher in d IL-4, e IFN-γ, and f IL-13 in the meninges after LPS injection in the McAb-proB group as compared to IgG control. n = 5 in each group. All experiments were performed at least in triplicate. Data were analyzed by unpaired T test, * P

Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

Techniques: Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Expressing