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