antibody against probdnf  (Santa Cruz Biotechnology)

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: Immunoblotting was performed using antibodies against proBDNF (Biosensis, R-176, polyclonal rabbit antibody, 0.25 μg/ml), CD42b (Santa Cruz, sc-59051, monoclonal mouse antibody, clone PM6/40, 1 μg/ml), or sortilin/NT3 (Abcam, ab16640, polyclonal rabbit antibody, 2 μg/ml).

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

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pro-BDNF Contributes to Hypoxia/Reoxygenation Injury in Myocardial Microvascular Endothelial Cells: Roles of Receptors p75 NTR and Sortilin and Activation of JNK and Caspase 3

doi: 10.1155/2018/3091424

Figure Lengend Snippet: Effects of the anti-pro-BDNF antibody on apoptosis, migration, and capillary-like-structure formation among MMECs after exposure to HG and H/R. (a) Effects of pro-BDNF on apoptosis were analyzed by flow cytometry of MMECs after different treatments: control, H/R, H/R + anti-pro-BDNF, and H/R + vehicle. (b, c) The functional effects of pro-BDNF on MMECs were assessed by capillary-like-structure formation and cell scratch assays. (d) Relative apoptosis levels and fold changes are expressed in relation to the control group. The H/R group showed markedly increased relative apoptosis levels, decreased capillary-like-structure formation, and reduced cell migration when compared with the control group. These effects were reversed by treatment with the anti-pro-BDNF antibody to the levels similar to those in the control group. The data were subjected to one-way ANOVA. The error bars represent SEM. ∗ P < 0.05 as compared with the control group; # P < 0.05 as compared with the H/R group or the H/R + vehicle group.

Article Snippet: After blocking in 5% fat-free dry milk, antibodies against pro-BDNF (Alomone, 1 : 400), BDNF (Abcam, 1 : 500), P75 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 100), sortilin (Abcam, 1 : 500), JNK (Santa Cruz Biotechnology, 1 : 100), cleaved caspase 3 (Asp175, Cell Signaling Technology, 1 : 1000), caspase 3, and β -actin (Santa Cruz Biotechnology) were employed.

Techniques: Migration, Flow Cytometry, Control, Functional Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pro-BDNF Contributes to Hypoxia/Reoxygenation Injury in Myocardial Microvascular Endothelial Cells: Roles of Receptors p75 NTR and Sortilin and Activation of JNK and Caspase 3

doi: 10.1155/2018/3091424

Figure Lengend Snippet: Effects of the anti-pro-BDNF antibody on the expression of p75 NTR and sortilin and apoptosis-related proteins. (a) Representative immunofluorescent images of pro-BDNF (red, first column), p-JNK (green, second column), BDNF (red, fourth column), JNK (green, fifth column), sortilin (red, seventh column), and p75 NTR (green, eighth column) in groups control, H/R, H/R + anti-pro-BDNF, and H/R + vehicle. (b–e) Representative Western blots and quantitative analysis of pro-BDNF, BDNF (b), p75 NTR , sortilin (c), JNK, p-JNK (d), caspase 3 and cleaved-caspase 3 expression (e) in response to different treatments. All the data were normalized to β -actin, and fold changes are expressed in relation to the control group. Exposure of MMECs to H/R resulted in significantly higher expression levels of pro-BDNF, p75 NTR , and sortilin and in activation of JNK and caspase 3 as compared with MMECs maintained under normal conditions (control). Nonetheless, there were no significant differences in BDNF, JNK, and caspase 3 expression levels after H/R. Treatment with the anti-pro-BDNF antibody significantly reversed the increase in the protein expression of pro-BDNF, p75 NTR , sortilin, p-JNK, and cleaved caspase 3 in MMECs after exposure to HG and H/R (H/R + anti-pro-BDNF). The data were subjected to one-way ANOVA. The error bars represent SEM. ∗ P < 0.05 as compared with the control group; # P < 0.05 as compared with the H/R or H/R + vehicle group.

Article Snippet: After blocking in 5% fat-free dry milk, antibodies against pro-BDNF (Alomone, 1 : 400), BDNF (Abcam, 1 : 500), P75 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 100), sortilin (Abcam, 1 : 500), JNK (Santa Cruz Biotechnology, 1 : 100), cleaved caspase 3 (Asp175, Cell Signaling Technology, 1 : 1000), caspase 3, and β -actin (Santa Cruz Biotechnology) were employed.

Techniques: Expressing, Control, Western Blot, Activation Assay