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, Control, Comparison, Staining, Adjuvant, 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, Comparison, Over Expression, Western Blot, Control, 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, Control, Comparison

Journal: Aging and disease

Article Title: Aging-Induced Brain-Derived Neurotrophic Factor in Adipocyte Progenitors Contributes to Adipose Tissue Dysfunction

doi: 10.14336/ad.2019.0810

Figure Lengend Snippet: Figure 5. proBDNF treatment-induced apoptosis/necroptosis of adipocytes required sortilin expression. (A) Immunoblot analysis of sortilin expression and apoptosis/necroptosis markers in adipocytes differentiated from C3H10T1/2 cells. (B) Immunoblot analysis of sortilin in adipocytes differentiated from C3H10T1/2 treated with siRNA or scrambled sequence controls (negative controls) (mean ± SEM; n = 4, *** p < 0.001). (C) Immunoblot analysis of cell surface protein detection in adipocytes differentiated from C3H10T1/2 cells treated with vehicle or pro-BDNF (10ng/ml) for 24 h (n = 4, means ± SEM, *** p < 0.001). Full images of Western blots are shown in supplementary Fig. 5.

Article Snippet: Fully differentiated adipocytes were exposed to DMEM supplemented with 10% FBS overnight and then treated with pro-BDNF (10ng/ml, Alomone Labs) [27, 28].

Techniques: Expressing, Western Blot, Sequencing

Journal: bioRxiv

Article Title: CNTF specifically slows down the axonal transport of signalling endosomes

doi: 10.1101/2025.10.09.681259

Figure Lengend Snippet: A) Male and female ChAT.eGFP mice were allocated into one of five groups: i) PBS (control); ii) CNTF; iii) proBDNF; iv) HGF; and v) NTRN. B) Each mouse received intramuscular injections of H C T combined with a single NTF into the left tibialis anterior and right soleus muscles to target fast and slow motor neurons, respectively. After a 4-8 h incubation period, time-lapse microscopy was performed on both sciatic nerves. C) H C T-labelled signalling endosomes (pseudo-coloured in magenta) from single ChAT.eGFP motor axons were individually tracked to quantify retrograde transport dynamics. Three representative retrogradely transported signalling endosomes are identified by yellow, cyan, and peach arrowheads connected by dashed lines across frames. Grey arrowheads and dashed lines identify a stationary endosome. See also Video 1 . Scale bar = 5 μm, frame interval = 3 s.

Article Snippet: Mice were divided into five experimental cohorts, and received 5-7 μg of H C T mixed with: a) phosphate buffered saline (PBS) as the control; b) 50 ng recombinant human CNTF protein (Peprotech, 450-13), c) 50 ng recombinant mouse cleavage-resistant proBDNF (Alomone labs, B-243), d) 25 ng recombinant human HGF (Bio-Techne, 294-HG[CF]), or e) 50 ng recombinant human NTRN (Peprotech, 450-11), as summarised in .

Techniques: Control, Muscles, Incubation, Time-lapse Microscopy

Journal: bioRxiv

Article Title: CNTF specifically slows down the axonal transport of signalling endosomes

doi: 10.1101/2025.10.09.681259

Figure Lengend Snippet: In both fast motor neurons (FMNs) and slow motor neurons (SMNs), proBDNF did not alter A) mean endosome speed ( p = 0.06 for motor neuron type; p = 0.19 for stimulation factor; p = 0.70 for interaction), B) maximum endosome speed ( p = 0.30 for motor neuron type; p = 0.62 for stimulation factor; p = 0.25 for interaction) or C) pausing percentage ( p = 0.24 for motor neuron type; p = 0.92 for stimulation factor; p = 0.34 for interaction). D ) Violin plots of individual endosomes show comparable distributions in all conditions (FMNs: PBS mean = 2.78 µm/s ± 0.03 [n = 495], proBDNF mean = 2.67 µm/s ± 0.03 [n = 496]; SMNs: PBS mean = 2.57 µm/ s ± 0.03 [n = 495], proBDNF mean = 2.53 µm/s ± 0.03 [n = 477]). Overlapping endosome frame-to-frame and mean endosome speed distribution curves confirm that proBDNF does not modulate retrograde transport in E) FMNs or F) SMNs. Statistical analyses were performed using two-way ANOVA and Holm-Šídák 1 s multiple comparisons tests. ns, not significant. n = 7-8. Black circles = males (n = 4 PBS; n = 2 CNTF), white circles = females (n = 4 PBS; n = 5 proBDNF).

Article Snippet: Mice were divided into five experimental cohorts, and received 5-7 μg of H C T mixed with: a) phosphate buffered saline (PBS) as the control; b) 50 ng recombinant human CNTF protein (Peprotech, 450-13), c) 50 ng recombinant mouse cleavage-resistant proBDNF (Alomone labs, B-243), d) 25 ng recombinant human HGF (Bio-Techne, 294-HG[CF]), or e) 50 ng recombinant human NTRN (Peprotech, 450-11), as summarised in .

Techniques:

Journal: Scientific reports

Article Title: Cognitive and sensorimotor benefits of moderate- and high-intensity exercise are associated with specific expression of neurotrophic markers in older rats.

doi: 10.1038/s41598-025-90719-4

Figure Lengend Snippet: Fig. 1. Experimental protocol. All trained animals carried out 4 weeks of training, 5 times per week either in MICT or HIIT. All animals performed both incremental test and grip strength at PRE, 2W and POST, while they only performed adhesive removal test and the novel object recognition test at the end of the training (in POST). During the incremental test, blood lactate was collected at every level to determine the SLT. At the end of the protocol (48 h after the last incremental test) both hippocampi and cortices were removed to perform Western blot and ELISA measurements.

Article Snippet: Quantification of pro and mature BDNF standard and cortical samples was respectively performed with proBDNF Rapid ELISA Kit (Biosensis®, BEK-2217-2P – sandwich ELISA–Thebarton, SA, Australia) and mBDNF Rapid ELISA Kit Kit (Biosensis®, BEK-2211-1P/2P–sandwich ELISA–Thebarton, SA, Australia) in the concentrated solutions following the manufacturer’s protocol.

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

Journal: Scientific reports

Article Title: Cognitive and sensorimotor benefits of moderate- and high-intensity exercise are associated with specific expression of neurotrophic markers in older rats.

doi: 10.1038/s41598-025-90719-4

Figure Lengend Snippet: Fig. 4. Effect of MICT and HIIT programs on cortical protein levels. (a) Level of cortical mBDNF (n = 19) measured by ELISA (in pg/ml-1) in MICT, HIIT and Control groups at POST training. Level of cortical (b) TrkB (n = 20), (c) pTrkB (n = 20), (d), p757NTR (n = 19), (e) NKCC1/KCC2 ratio (n = 19), (f) KCC2 (n = 18) and (g) CTSB (n = 20) in MICT, HIIT and Control groups at POST training. Protein level measured by Western blot, was calculated as the ratio of the protein of interest divided by the protein of normalization relative to the control. The normalization protein was the α-tubulin for all the molecules except for the KCC2 which was β3-tubulin. Pictures of Western blot membranes are shown below each graph. Dashed lines were added to separate the groups. Note that the α-tubulin used for TrkB (c) was the same used for pTrkB (d), as membranes were stripped between these two quantifications. The samples derived from the same experiment and that gels/ blots were systematically processed in parallel the same day (2 gels per protein to reach a sufficient number of samples). *Significant differences in protein levels between groups. Data is expressed in mean ± SD (a–f) and in median (min to max) (g).

Article Snippet: Quantification of pro and mature BDNF standard and cortical samples was respectively performed with proBDNF Rapid ELISA Kit (Biosensis®, BEK-2217-2P – sandwich ELISA–Thebarton, SA, Australia) and mBDNF Rapid ELISA Kit Kit (Biosensis®, BEK-2211-1P/2P–sandwich ELISA–Thebarton, SA, Australia) in the concentrated solutions following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Derivative Assay

Journal: Scientific reports

Article Title: Cognitive and sensorimotor benefits of moderate- and high-intensity exercise are associated with specific expression of neurotrophic markers in older rats.

doi: 10.1038/s41598-025-90719-4

Figure Lengend Snippet: Fig. 5. Effect of MICT and HIIT programs on hippocampal protein levels. (a) Level of hippocampal IGF- 1(n = 21) measured by ELISA in MICT, HIIT and Control groups at POST training. Level of hippocampal (b) IGFB-2 (n = 21), (c) p75NTR (n = 22), (d) TrkB (n = 21), (e) CTSB (n = 22), (f) NKCC1 (n = 21), (g) KCC2 (n = 20), and (h) the NKCC1/KCC2 ratio (n = 18) measured by Western blot in MICT, HIIT and Control groups at POST. Protein level was calculated as the ratio of the protein of interest divided by the protein of normalization relative to the control. The normalization protein was the α-tubulin for all the molecules except for the KCC2 which was β3-tubulin. Pictures of the Western blot membranes are shown below each graph. Dashed lines were added to separate the groups. Note that the α-tubulin used for p75NTR (c) was the same used for NKCC1 (f) as their molecular weight are different, the quantification of both was conducted on the same membranes. The samples derived from the same experiment and that gels/blots were processed in parallel the same day (2 gels per protein to reach a sufficient number of samples). *Significant differences in protein levels between groups. All data is expressed in mean ± SD.

Article Snippet: Quantification of pro and mature BDNF standard and cortical samples was respectively performed with proBDNF Rapid ELISA Kit (Biosensis®, BEK-2217-2P – sandwich ELISA–Thebarton, SA, Australia) and mBDNF Rapid ELISA Kit Kit (Biosensis®, BEK-2211-1P/2P–sandwich ELISA–Thebarton, SA, Australia) in the concentrated solutions following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Molecular Weight, Derivative Assay

Journal: Theranostics

Article Title: A Novel Peptide Interfering with proBDNF-Sortilin Interaction Alleviates Chronic Inflammatory Pain

doi: 10.7150/thno.29703

Figure Lengend Snippet: Mapping of proBDNF-sortilin interaction interface. (A) Schematic diagram illustrating the design of human BDNF prodomain variants. Six variants with a series of amino acids deletions were used in this study. V66M, Val66Met; S, Signal peptide (amino acid 1-18); BDNF, mature BDNF (amino acid 129-247); HA, C-terminal HA epitope. (B) Immunoblots of co-immunoprecipitation (Left) or inputs (Right) of sortilin and proBDNF variants obtained with antibody against HA or Myc epitope. IP, Immunoprecipitation; IB, Immunoblot. (C) Left , live fluorescence micrograph of BiFC mapping assay (Yellow) using HEK293T cells with VC155-sortilin (Sort1) and VN155(I152L)-BDNF prodomain variants. Scale bar represents 50 µm. Right , quantification of fluorescence intensity of BiFC assay. P <0.0001, one-way ANOVA. ** P <0.01 versus Fl group; *** P <0.0001 versus Fl group; ## P <0.001 versus variant I; ### P <0.0001 versus variant I group; n.s., non-significant. Values are means ± SEM. n = 100 individual cells from 4 images. (D) Left , representative immunoblots of supernatant and cell lysates of HEK293 cells overexpressing sortilin and BDNF prodomain variants obtained with antibodies against HA. Right , densitometry analyses of secreted BDNF in supernatants, normalized to input in lysates. P <0.001, one-way ANOVA. * P <0.05, versus Fl group; *** P <0.001, versus Fl group; ## P <0.05, versus I group. Values are means ± SEM. n = 4. IB, Immunoblot. (E) BDNF ELISA quantification of supernatant collected from HEK293 cells overexpressing sortilin and BDNF prodomain variants. P <0.0001, one-way ANOVA. * P <0.05; ** P <0.01; *** P <0.001, versus Sort1/Fl group; ## P <0.05, versus Sort1/variant I group. Values are means ± SEM. Each data point represents the average of 3 independent experiments.

Article Snippet: The primary antibodies used included anti-proBDNF antibody (#ANT-006-AG, Alomone Labs Ltd, Jerusalem, Israel), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell signalling Technology, Danvers, MA, USA), anti-NeuN antibody (MAB377, EMD Millipore Corporation, Billerica, MA, USA) and anti-sortilin antibody (Abcam, Cambridge, UK).

Techniques: Western Blot, Immunoprecipitation, Fluorescence, Mapping Assay, Bimolecular Fluorescence Complementation Assay, Variant Assay, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: A Novel Peptide Interfering with proBDNF-Sortilin Interaction Alleviates Chronic Inflammatory Pain

doi: 10.7150/thno.29703

Figure Lengend Snippet: Construction of a novel peptide targeting proBDNF-sortilin Interaction. (A) Schematic diagram illustrating the design of a series of blocking peptides targeting proBDNF-sortilin interaction interface. Scr-bdnf84-83 (scr), scrambled peptide with permutation of peptide sequence at amino acid 84-93. (B) Immunoblots of co-immunoprecipitation (upper) or reverse co-immunoprecipitation (lower) of sortilin and proBDNF variants in cell lysates overexpressing full-length proBDNF and sortilin followed by peptide incubation obtained with antibodies against HA or Myc. IP, Immunoprecipitation; IB, Immunoblot. (C) Schematic diagram of the design of the Tat-tagged interfering peptide. Scr-bdnf89-98-Tat, Tat-tagged scrambled peptide with permutation of peptide sequence at amino acid 89-98. (D) Live fluorescence micrographs of 5FAM (green) or bright field in rat DRG neuronal cultures before (0 min) and incubated with 5 µM 5FAM-tagged bdnf89-98-Tat (bdnf-Tat) for 2 h. Scale bar represents 150 µm. (E) Fluorescence micrograph of 5FAM (green), sortilin (red) or DAPI (blue) in rat DRG neuronal cultures with peptides pre-treatment for 30 min. Scale bars represent 25 µm. (F) Left , fluorescence micrograph of BiFC assay (yellow) of HEK293T cells overexpressing full-length BDNF and sortilin with vehicle, 10 µM scr-Tat or bdnf-Tat pre-treatment for 30 min. Scale bars represent 25 µm. Right , Determination of the corrected total cell fluorescence (CTCF) of the BiFC signal. P <0.0001 , one-way ANOVA. *** P <0.0001, scr-Tat treatment versus bdnf-Tat treatment. Mean values ± SEM. n = 100 individual cells from 5 images. (G) Effect of 30 min peptide pre-treatment on activity-dependent secretion of BDNF in rat DRG neuronal cultures with high-potassium stimulus using ELISA. P <0.05, one-way ANOVA. * P <0.05, vehicle versus scr-Tat treatment or scr-Tat treatment versus bdnf-Tat treatment. Values are means ± SEM. Each data point represents the average of 3 independent experiments.

Article Snippet: The primary antibodies used included anti-proBDNF antibody (#ANT-006-AG, Alomone Labs Ltd, Jerusalem, Israel), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell signalling Technology, Danvers, MA, USA), anti-NeuN antibody (MAB377, EMD Millipore Corporation, Billerica, MA, USA) and anti-sortilin antibody (Abcam, Cambridge, UK).

Techniques: Blocking Assay, Sequencing, Western Blot, Immunoprecipitation, Incubation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay