Journal: Molecular Brain

Article Title: proBDNF is modified by advanced glycation end products in Alzheimer’s disease and causes neuronal apoptosis by inducing p75 neurotrophin receptor processing

doi: 10.1186/s13041-018-0411-6

Figure Lengend Snippet: proBDNF modified by MGO induces apoptosis in control mice differentiated hippocampal neural stem cells . a) Representative pictures of neuronal primary culture treated for 6 days (7DIV) with: untreated (control), MGO modified BSA (MBSA), mBDNF, proBDNF and two doses of MGO modified proBDNF (MproBDNF). DAPI staining (white) of nuclei shows apoptotic morphology (white arrow). Immunostaining with anti βIIItubulin (red) show differentiation degree. b) Bars represent the quantification of apoptosis (by nuclei morphology), expressed as the mean of % apoptosis ± SD of the total nuclei. c) The quantification of differentiation is expressed as the mean of % differentiation (βIIItubulin immunoreactivity quantified by ImageJ, divided by the number of β III tubulin positive cells). Values represent the mean of three independent experiments. Scale bar =25 μm . ** p < 0,01, *** p < 0,001 two tailed Student’s “t” test

Article Snippet: Recombinant human proBDNF (Alomone) was modified by the reactive carbonyl specie MGO that react with free amino groups of Lys residues on proteins, leading to the formation of CEL adducts and intermolecular crosslinks [ ].

Techniques: Modification, Staining, Immunostaining, Two Tailed Test, T-Test

Journal: Molecular Brain

Article Title: proBDNF is modified by advanced glycation end products in Alzheimer’s disease and causes neuronal apoptosis by inducing p75 neurotrophin receptor processing

doi: 10.1186/s13041-018-0411-6

Figure Lengend Snippet: Human proBDNF from CSF AD patients induces apoptosis in neuronal culture. a) Representative pictures of neuronal primary culture from control mice Dentate Gyrus. Cells were treated for 6 days (7DIV) with human CSF from controls (control CSF) and from AD-affected patients (ADCSF). DAPI staining of nuclei shows apoptotic morphology (white arrow). Immunofluorescence with anti p75 ICD domain (green) and anti Sortilin (red). Inset indicate a higher magnification image of the boxed areas. White arrow in control CSF shows neurons with peripheral p75ICD location. White arrows in AD CSF show p75ICD nuclear location. b) Representative image of western blot showing the levels of proBDNF in CSF and in CSF immunodepleted with anti proBDNF (ID), from control and AD samples. c and d) Cells are treated with human 8 control CSF and 8 ADCSF, in both cases directly (CSF) or proBDNF immunodepleted (SCFID). c) Bars represent the quantification of apoptosis (by nuclei morphology), expressed as the mean of % apoptosis ± SD of the positive cells for both p75 and Sortilin. d) Bars represent the quantification of the mean of the % ± SD of neurons with the ICDp75 translocated to the nuclei (% ICD nuclear). Scale bar =50 μm . * p < 0,05, ** p < 0,01, two tailed Student’s “t” test

Article Snippet: Recombinant human proBDNF (Alomone) was modified by the reactive carbonyl specie MGO that react with free amino groups of Lys residues on proteins, leading to the formation of CEL adducts and intermolecular crosslinks [ ].

Techniques: Staining, Immunofluorescence, Western Blot, Two Tailed Test, T-Test

Journal: Molecular Brain

Article Title: proBDNF is modified by advanced glycation end products in Alzheimer’s disease and causes neuronal apoptosis by inducing p75 neurotrophin receptor processing

doi: 10.1186/s13041-018-0411-6

Figure Lengend Snippet: Apoptosis and p75 processing are increased in neuronal cultures from APP/PS1 animals. Primary cultures of differentiated mouse hippocampal NSCs were treated for 6 days (7DIV) with MGO modified BSA (MBSA) or with MGO modified proBDNF (MproBDNF). a) Representative immunofluorescence pictures of cells from WT and APP/P1 mice labeled with ICDp75 (green), βIIItubulin (red) and DAPI (white). Inset represents a higher magnification image of the boxed areas indicating citoplasmatic (WT) and nuclear localization (APP/PS1) of ICDp75. b) Bars represent the quantification of apoptosis (by nuclei morphology), expressed as the mean of % apoptosis ± SD of the positive cells for both ICDp75 and βIIItubulin. c) Bars represent the mean of the % ± SD of neurons with the ICDp75 translocated to the nuclei (% ICD nuclear). The values represent the mean of three independent experiments Scale bar =50 μm . ** p < 0,01, two tailed Student’s “t” test

Article Snippet: Recombinant human proBDNF (Alomone) was modified by the reactive carbonyl specie MGO that react with free amino groups of Lys residues on proteins, leading to the formation of CEL adducts and intermolecular crosslinks [ ].

Techniques: Modification, Immunofluorescence, Labeling, Two Tailed Test, T-Test

Journal: Frontiers in Immunology

Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF

doi: 10.3389/fimmu.2020.575607

Figure Lengend 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.

Article Snippet: Platelets and neuroblastic cells were then incubated at RT with mouse anti-human proBDNF antibody (R&D System, mab31751, monoclonal mouse antibody, clone 584412, diluted 1:25 or Biosensis, R-176 polyclonal rabbit antibody diluted 1:25) or mouse IgG 2b /rabbit isotype control (R&D System, MAB004 and AB-105-C diluted 1:25) for 30 min. Alexa Fluor 488 or 647 conjugated donkey anti-mouse or rabbit secondary antibody (Invitrogen, diluted 1:100) was added for 30 min at RT in the dark.

Techniques: Western Blot, Recombinant, Molecular Weight, Control, Membrane, Incubation, Flow Cytometry, Expressing, Confocal Microscopy, Imaging, Staining, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF

doi: 10.3389/fimmu.2020.575607

Figure Lengend 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<0.05. (E, F) Correlation between BDNF and proBDNF molar concentrations in human platelets (E) and in plasma (F) . Dotted lines represent 95% confidence intervals, n=20 participants (#1 to #20).

Article Snippet: Platelets and neuroblastic cells were then incubated at RT with mouse anti-human proBDNF antibody (R&D System, mab31751, monoclonal mouse antibody, clone 584412, diluted 1:25 or Biosensis, R-176 polyclonal rabbit antibody diluted 1:25) or mouse IgG 2b /rabbit isotype control (R&D System, MAB004 and AB-105-C diluted 1:25) for 30 min. Alexa Fluor 488 or 647 conjugated donkey anti-mouse or rabbit secondary antibody (Invitrogen, diluted 1:100) was added for 30 min at RT in the dark.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF

doi: 10.3389/fimmu.2020.575607

Figure Lengend 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 < 0.05 vs . ctrl, n=20 participants (#1 to #20). Ctrl, control; ADP, adenosine diphosphate; TRAP, thrombin receptor-activating peptide; AA, arachidonic acid.

Article Snippet: Platelets and neuroblastic cells were then incubated at RT with mouse anti-human proBDNF antibody (R&D System, mab31751, monoclonal mouse antibody, clone 584412, diluted 1:25 or Biosensis, R-176 polyclonal rabbit antibody diluted 1:25) or mouse IgG 2b /rabbit isotype control (R&D System, MAB004 and AB-105-C diluted 1:25) for 30 min. Alexa Fluor 488 or 647 conjugated donkey anti-mouse or rabbit secondary antibody (Invitrogen, diluted 1:100) was added for 30 min at RT in the dark.

Techniques: Activation Assay, Clinical Proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: ProBDNF stably expressed in HEK293F cells is glycosylated. N-Glycosylation of proBDNF and the prodomain is documented by mass shift following deglycosylation with PNGase F: A, detection of proBDNF and BDNF by an antibody recognizing the BDNF region; B, detection of proBDNF and the prodomain by an antibody recognizing the prodomain region. The gel images were spliced as indicated by space to exclude samples not related to the study.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Extracted ion chromatograms (XIC) of (A) nonglycosylated, NYLDAANMSMR and (B) deglycosylated, NYLDAADMSMR glycopeptide of proBDNF secreted by HEK293F cells confirm site occupancy higher than 99%. C, tandem mass spectrum verifies deglycosylation of the proBDNF peptide under [18O]water.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Expression of wildtype (WT) proBDNF and its N121Q mutant (NQ) in stably transfected HEK293F cells. Protein expression was determined by Western blotting using an antibody, recognizing both BDNF and proBDNF, in the conditioned media (A) and cell lysates (B). Arrows point to (pro)BDNF forms: glycosylated proBDNF (black arrow), nonglycosylated proBDNF (white arrow), and mature BDNF (gray arrow). M indicates MagicMark molecular weight marker. Expression of mRNA was determined in WT and N121Q mutant by RT-PCR (C). ProBDNF/BDNF expression in cell lysates (D) or conditioned media (E) of HEK293F cells expressing N121Q mutant treated with proteasome inhibitor MG132 (MG), lysosomal inhibitor chloroquine (CQ), or chemical chaperone 4-phenylbutyric acid (PBA). The black arrow points to the position of nonglycosylated proBDNF.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Expressing, Mutagenesis, Stable Transfection, Transfection, Western Blot, Molecular Weight, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Kinetics of cleavage of glycosylated and nonglycosylated proBDNF by furin. Mass difference between glycosylated and nonglycosylated proBDNF enabled densitometric quantification of each proBDNF form: A, representative blot of the cleavage kinetic at the indicated time intervals; B, densitometric analysis of proBDNF intensities at the indicated time intervals. Results are expressed as percent of the proBDNF concentration (mean ± S.D., n = 3) at the beginning of the reaction. Note that proBDNF without C-terminal Myc-FLAG tag was used in the reaction as described under “Experimental procedures.”

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Concentration Assay, FLAG-tag

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Mass spectra of proBDNF under the following conditions. A, precursor profile of the tryptic glycopeptide NYLDAANM(ox)SM(ox)R of proBDNF expressed in HEK293F cells; B, HCD fragmentation spectrum of the major glycoform (m/z 1132.42) at low collision energy (10% NCE) showing that characteristic LacdiNAc oxonium ion HexNAc-HexNAc (m/z 407.17) and its fucosylated form (m/z 553.22) with complementary y-ions (m/z 1373, 1381, 1454, and 1555) dominate the composition.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Relative abundance of the glycoforms of HEK293F-produced proBDNF and prodomain expressed as % of all identified glycoforms

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Produced

Journal: The Journal of Biological Chemistry

Article Title: N -Glycosylation is required for secretion of the precursor to brain-derived neurotrophic factor (proBDNF) carrying sulfated LacdiNAc structures

doi: 10.1074/jbc.RA119.009989

Figure Lengend Snippet: Impact of modulators of glycosylation on the BDNF/proBDNF ratio in HEK293F cells. Western blotting using antibodies to prodomain and mature BDNF regions was analyzed by densitometry for the following: A, intracellular BDNF to proBDNF ratio; B, secreted BDNF to proBDNF ratio; C, intracellular prodomain to proBDNF ratio; and D, secreted prodomain to proBDNF ratio. The representative Western blotting is labeled as the graphs: CTRL, control; CHLOR, 50 mm sodium chlorate; KIF, 1 μg/ml kifunensine; TNMC, 5 μg/ml of tunicamycin. Results are expressed as scatter plot (mean ± S.D., n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with CTRL, one-way analysis of variance with Bonferroni adjustment.

Article Snippet: Glycosylated human proBDNF without C-terminal Myc-FLAG tags produced in HEK293F cells and nonglycosylated human proBDNF produced in E. coli (Alomone Labs, number B-257) were used to study the kinetic of cleavage by furin (R&D Systems, number 1503-SE-010).

Techniques: Western Blot, Labeling

Journal: Scientific Reports

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

doi: 10.1038/srep27171

Figure Lengend Snippet: ( 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 < 0.05, **p < 0.01 versus control, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). ( B ) H&E staining (a and d) and immunohistochemsitry (b,c and e–g) of proBDNF in the foot skin at 3 h post-formalin injection. proBDNF is expressed in the epidermis, basal layer and subcutaneous layers in the foot skin (b,c); Higher magnification (box in b) showing proBDNF is also mildly expressed in the nerve fibers in the control plantar (c); Responding to peripheral inflammation by 5% formalin intra-plantar injection, intensive proBDNF immunoreactivity is observed and mainly localized in the inflammatory cells (f, black arrows) and nerve fiber-like structures (g). Scale bars: 50 μm, 3 replicates, n = 3 per group. ( C ) a, Representative Western blot of proBDNF and mBDNF; b–d, Semi-quantitative analyses of mBDNF, proBDNF and their ratio in the inflamed tissue after Complete Freund Adjuvant (CFA, 10 μL) intra-plantar injection into Kunming mice (*p < 0.05, **p < 0.01versus control, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). ( D ) Histological staining (a) and proBDNF immunohistochemistry (b,c) in the plantar at 1 day post-CFA injection; c, higher magnification of box in b showing that proBDNF is highly expressed in the inflammatory cells. Scale bar, 100 μm, 3 replicates, n = 3 per group. Data bars represent mean ± s.e.m.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Western Blot, Injection, Staining, Immunohistochemistry

Journal: Scientific Reports

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

doi: 10.1038/srep27171

Figure Lengend Snippet: ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P < 0.05, **p < 0.01 versus baseline, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 10–12 per group). ( B ) Ectopic overexpression of proBDNF by intra-plantar injection of Ad-proBDNF or Ad-EGFP reduces PWT dramatically in Kunming mice. (a) Representative proBDNF Western blot (upper panel) and its semi-quantitative analysis (lower panel, *P < 0.05, ***p < 0.001 versus control, ### p < 0.001 versus indicated groups, one-way ANOVA followed by Tukey’s Multiple Comparison post hoc test, n = 4 per group); (b) Representative fluorescent images after delivery of Ad-EGFP . Scale bar, 50 μm, 3 replicates, n = 3 per group; (c) PWT at 7 days post-injection of Ad-proBDNF or Ad-EGFP control (***p < 0.001 versus Ad-EGFP , student’s t test, n = 7 per group). ( C ) Co-injection of proBDNF (0.1 μg), but not mBDNF (0.1 μg) restored the biphasic nociceptive response after low-concentration of formalin (0.5%) intra-plantar injection (*p < 0.05versus vehicle, two-way ANOVA followed by Bonferroni’s Multiple Comparison post hoc test, n = 10–12 per group). ( D ) Exogenous proBDNF (1 μg) intra-plantar injection induces ERK activation in the ipsilateral spinal cord dorsal horn at 3 h post-injection, Scale bar, 50 μm, n = 3 per group, 3 replicates. ( E ) Spinal p-ERK expression at 3 h after proBDNF (1 μg) intra-plantar injection. (a) Representative Western blot and (b,c) their semi-quantitative analyses of p-ERK, (*p < 0.05, **p < 0.01 versus proBDNF-R, one-way ANOVA followed by Dunnett’s Multiple Comparison post hoc test, n = 3 per group). Data bars represent mean ± s.e.m.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

Techniques: Injection, Over Expression, Western Blot, Concentration Assay, Activation Assay, 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: ( 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 < 0.001 versus control, # p < 0.05 versus indicated group, one-way ANOVA followed by Tukey’s Multiple Comparison post hoc test). Neurospheres treated with proBDNF (100 ng/mL) showed dormancy without any neuronal migration and neurospheres had no morphological changes. Neurospheres treated with 2B11 (100 ng/ml) showed strong migration capability comparing with other groups. Neurospheres treated with 2B11 and proBDNF (100 ng/ml) showed similar ability of migration with sheep anti-proBDNF antibody treatment group.

Article Snippet: For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C.

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