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: Brain Sciences

Article Title: Ouabain Counteracts Retinal Ganglion Cell Death Through Modulation of BDNF and IL-1 Signaling Pathways

doi: 10.3390/brainsci15020123

Figure Lengend Snippet: Primary and secondary antibodies for Western blotting.

Article Snippet: Mouse anti-proBDNF , Santa Cruz Biotechnology (Dallas, TX, USA) , 1:1000.

Techniques: Western Blot

Journal: Experimental Neurobiology

Article Title: Astrocytic proBDNF and Tonic GABA Distinguish Active versus Reactive Astrocytes in Hippocampus

doi: 10.5607/en.2018.27.3.155

Figure Lengend Snippet: The expression of proBDNF and mBDNF in resting astrocytes of hippocampus. (A) Representative confocal images for the immunostained proBDNF in CA1 hippocampus of GFAP-GFP mice (red; proBDNF, green; GFP). (B) Bar graph showing the normalized average intensity of astrocytic and non-astrocytic proBDNF in GFAP-GFP mice. Unpaired two-tailed-t-test ( *** p<0.001) (n=34 for Astrocyte; n=34 for Neuron). (C) Representative confocal images for the immunostained mBDNF in CA1 hippocampus of GFAP-GFP mice (red; mBDNF, green; GFP). (D) Bar graph showing the normalized average intensity of astrocytic and non-astrocytic mBDNF in GFAP-GFP mice. Unpaired two-tailed-t-test ( **** p<0.0001) (n=36 for Astrocyte; n=35 for Neuron).

Article Snippet: Primary antibodies used are as following: chicken anti-GFAP (1:500, ab5541, Millipore), guinea pig anti-GABA (1:200, ab175, Millipore), chicken anti-proBDNF (1:200, ab9042, Millipore, epitope; fusion protein from mouse proBDNF containing only the prodomain region), rabbit anti-BDNF (1:50, sc20981, Santacruz, epitope; recombinant BDNF containing amino acid 130-247 from N-terminus).

Techniques: Expressing, Two Tailed Test

Journal: Experimental Neurobiology

Article Title: Astrocytic proBDNF and Tonic GABA Distinguish Active versus Reactive Astrocytes in Hippocampus

doi: 10.5607/en.2018.27.3.155

Figure Lengend Snippet: The level of proBDNF is enhanced in EE. (A) Expression of proBDNF in GFAP-positive astrocytes at molecular layer of DG hippocampus (red; proBDNF, green; GFAP). (B) Experimental timeline for histology in enriched environment (EE). (C) Representative confocal images for the immunostained proBDNF (red; proBDNF, blue; DAPI) in DG hippocampus in EE or SC mice. (D) Bar graph showing the comparison of the average intensity of proBDNF immunoreactivity in molecular layer of DG. Unpaired two-tailed-t-test ( *** p<0.001) (n=16 for SC; n=21 for EE).

Article Snippet: Primary antibodies used are as following: chicken anti-GFAP (1:500, ab5541, Millipore), guinea pig anti-GABA (1:200, ab175, Millipore), chicken anti-proBDNF (1:200, ab9042, Millipore, epitope; fusion protein from mouse proBDNF containing only the prodomain region), rabbit anti-BDNF (1:50, sc20981, Santacruz, epitope; recombinant BDNF containing amino acid 130-247 from N-terminus).

Techniques: Expressing, Two Tailed Test

Journal: Experimental Neurobiology

Article Title: Astrocytic proBDNF and Tonic GABA Distinguish Active versus Reactive Astrocytes in Hippocampus

doi: 10.5607/en.2018.27.3.155

Figure Lengend Snippet: The level of proBDNF is not increased in STAB. (A) Representative confocal images for the immunostained proBDNF (red; proBDNF, green; GFAP, magenta; DiI indicating injury site) in CA1 hippocampus of STAB mice. (B) Bar graph showing the comparison of the average intensity of proBDNF immunoreactivity in GFAP-positive pixels in STAB mice. One-way ANOVA with Tukey's multiple comparison test ( **** p<0.0001, NS; non-significant, p>0.05) (n=37 for contra; n=49 for penumbra; n=55 for core).

Article Snippet: Primary antibodies used are as following: chicken anti-GFAP (1:500, ab5541, Millipore), guinea pig anti-GABA (1:200, ab175, Millipore), chicken anti-proBDNF (1:200, ab9042, Millipore, epitope; fusion protein from mouse proBDNF containing only the prodomain region), rabbit anti-BDNF (1:50, sc20981, Santacruz, epitope; recombinant BDNF containing amino acid 130-247 from N-terminus).

Techniques:

Journal: Journal of Inflammation Research

Article Title: Up-Regulation of ProBDNF/p75 NTR Signaling in Spinal Cord Drives Inflammatory Pain in Male Rats

doi: 10.2147/JIR.S387127

Figure Lengend Snippet: Schematic diagram shows that peripheral inflammation upregulates proBDNF and p75 NTR in the spinal cord dorsal horn, which activate the downstream signaling ERK1/2 and NFκB, thereby promoting the spinal neuroinflammation and contributing to inflammatory pain. The administration of Ab-proB or p75 NTR -ECD ameliorates inflammatory pain by suppressing ERK1/2-NFκB pathway.

Article Snippet: Without washing, the sections were incubated with the primary antibodies (anti-proBDNF) as described previously, anti-p75NTR (catalog: #8238, rabbit monoclonal, 1:1000, Cell Signaling Technology), anti-GFAP (catalog: SAB5201104, mouse monoclonal, 1:1000, Millipore), anti-iba-1 (catalog: ab178846, rabbit monoclonal, 1:1000, Abcam), anti-iba-1 (catalog: ab178846, goat monoclonal, 1:500, Abcam), anti-NeuN (catalog: #94403, rabbit monoclonal, 1:1000, Cell Signaling Technology), anti-p-ERK (catalog: #4370, rabbit monoclonal, 1:1000, Cell Signaling Technology), anti-phospho-p65 (catalog: #3033, 1:1000, Cell Signaling Technology) overnight at 4 °C.

Techniques:

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: Protein samples were separated on a 12% SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto a PVDF 0.2 μm membrane and fixed with glutaraldehyde 0.5% for 30 min. Membranes were washed three times with TBS-T (Tris-buffered saline, 0.1% Tween 20) for 10 min and blocked for 1 h at RT in blocking solution [3% bovine serum albumin (BSA) in TBS-T or 5% milk in TBS-T] before incubation with anti proBDNF or anti α 2 -macroglobulin antibodies (Biosensis, anti-proBDNF R-176 polyclonal rabbit antibody, 0.25 μg/ml; R&D systems, anti-proBDNF mab31751 monoclonal mouse antibody, clone 584412, 0.5 μg/ml; Abbexa, anti-α 2 -macroglobulin abx132389, monoclonal mouse antibody, 1 μg/ml) overnight at 4°C.

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

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: Mouse monoclonal anti-human proBDNF antibody (1:10,000, GeneCopoeia, Rockville, MD, number H10001G-MA) recognizing the prodomain region, was used to detect the proBDNF and prodomain ( 10 ).

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

Article Snippet: Mouse monoclonal anti-human proBDNF antibody (1:10,000, GeneCopoeia, Rockville, MD, number H10001G-MA) recognizing the prodomain region, was used to detect the proBDNF and prodomain ( 10 ).

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: Mouse monoclonal anti-human proBDNF antibody (1:10,000, GeneCopoeia, Rockville, MD, number H10001G-MA) recognizing the prodomain region, was used to detect the proBDNF and prodomain ( 10 ).

Techniques: Western Blot, Labeling