Journal: PLoS ONE

Article Title: S100B Protein, Brain-Derived Neurotrophic Factor, and Glial Cell Line-Derived Neurotrophic Factor in Human Milk

doi: 10.1371/journal.pone.0021663

Figure Lengend Snippet: A: BDNF band at approximately 27 kDa. B: GDNF band at approximately 20 kDa.

Article Snippet: Immunoblotting was performed using rabbit BDNF and GDNF antibodies (Wuhan Boster Bioligical Technology.,LTD, China).

Techniques:

Journal: PLoS ONE

Article Title: S100B Protein, Brain-Derived Neurotrophic Factor, and Glial Cell Line-Derived Neurotrophic Factor in Human Milk

doi: 10.1371/journal.pone.0021663

Figure Lengend Snippet: M = DL500 DNA Marker. A: bands of β-actin at 256 bp. B: bands of S100B at 147 bp. C: bands of BDNF at 379 bp. D: bands of GDNF at 132 bp. The bands of cytokines from milks collected at day 3, 10, 30, 90 after parturition were not found to vary with time.

Article Snippet: Immunoblotting was performed using rabbit BDNF and GDNF antibodies (Wuhan Boster Bioligical Technology.,LTD, China).

Techniques: Marker

Journal: Cell Death & Disease

Article Title: Retrograde transport of neurotrophin receptor TrkB-FL induced by excitotoxicity regulates Golgi stability and is a target for stroke neuroprotection

doi: 10.1038/s41419-025-07990-6

Figure Lengend Snippet: A – D Kinetics of TrkB-FL downregulation. Primary cortical cultures were treated with 100 μM NMDA and its co-agonist 10 μM glycine (hereafter referred to as ‘NMDA’). Immunofluorescence ( A ) and immunoblotting ( B ) used a C-terminal (C-ter) isoform-specific antibody (TrkB-FL Ct) recognizing both the full-length protein (FL) and the intracellular fragment (f32). A Shows TrkB-FL (green) and nuclei (blue, DAPI stain). Arrowheads indicate varicosities in neuronal projections. Scale bar: 20 μm. Insets show cell body details for untreated cells and cells treated with NMDA for 120 min. B Compares the decrease in TrkB-FL and formation of f32 with PSD-95 downregulation, detected using a C-terminal antibody (PSD-95 Ct). Calpain activation was confirmed by the accumulation of characteristic spectrin breakdown products (BDPs; 150 and 145 kDa). Neuron-specific enolase (NSE) served as a loading control for protein normalization. C , D Quantification of normalized TrkB-FL and PSD-95 levels, shown relative to levels in the absence of NMDA (control). Data are represented as means ± SD. Statistical analysis: one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (** P < 0.01, *** P < 0.001, **** P < 0.0001; 0-90 min, n = 5; 120 min, n = 3). E , F Effect of dynasore preincubation (80 μM, 30 min) on TrkB-FL levels after 2 h NMDA treatment. Data are means ± SD ( n = 4); statistical analysis as above (* P < 0.05). G Sequences of the cell-penetrating neuroprotective peptide (MTFL 457 ) and control peptide (MTMyc). Both contain Tat amino acids (aa) 47–57 (italic), followed by rat TrkB-FL aa 457-471 (light blue) or c-Myc aa 408-421 (dark blue), respectively. H , I Effect of peptide preincubation (25 μM, 30 min) on TrkB-FL shedding and processing during NMDA treatment. Culture media were analyzed using an antibody against the TrkB-FL extracellular domain (panTrkB), which recognizes the ectodomains (ECDs) of all isoforms (TrkB-ECD), to assess receptor shedding by metalloproteinase (MP) activation. Total lysates were analyzed with the TrkB-FL Ct antibody to evaluate TrkB-FL calpain processing via f32 production. Relative TrkB-ECD levels are shown as means ± SD (0-4 h, n = 7; 6 h, n = 4). Statistical analysis: two-way ANOVA followed by Bonferroni post hoc test (** P < 0.01, **** P < 0.0001, comparing each peptide + NMDA vs. peptide alone; # P < 0.05, #### P < 0.0001, comparing MTMyc vs. MTFL 457 at each time point). J , K Effect of NMDA on total and cell-surface pY816-TrkB-FL levels. Cultures were preincubated with peptides as above, then treated briefly with NMDA (1 h) to minimize receptor degradation. Cell-surface proteins were biotin-labeled and precipitated, then compared to corresponding total lysates. Data are represented as means ± SD ( n = 4). Statistical analysis: two-way ANOVA followed by Bonferroni post hoc test ( n.s . = n ot significant; ** P < 0.01, comparing NMDA vs. no NMDA; ## P < 0.01, comparing MTMyc + NMDA vs. MTFL 457 + NMDA).

Article Snippet: Primary antibodies against the following proteins were used: Hrs (Santa Cruz Biotechnology; Cat#sc-271455, RRID:AB_10648901), NSE (Millipore; Cat#AB951, RRID:AB_92390), spectrin alpha chain (Millipore; Cat#MAB1622, RRID:AB_94295), TrkB-FL C-ter (Santa Cruz Biotechnology; sc-11, RRID:AB_632554), TrkB extracellular sequences or panTrkB (Santa Cruz Biotechnology; sc-136990, RRID:AB_2155262), PSD-95 C-ter (Transduction Laboratories; Cat#610496, RRID:AB_2315218), pY816-TrkB-FL (Boster; Cat# P01388 ).

Techniques: Immunofluorescence, Western Blot, Staining, Activation Assay, Control, Labeling

Journal: The Journal of Neuroscience

Article Title: Transient Growth Factor Delivery Sustains Regenerated Axons after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1903-07.2007

Figure Lengend Snippet: Plasmid map of the tetracycline-regulatable retroviral expression vector pLN–tet-on and timeline of in vivo experiments. A, Expression of the reversed tetracycline transactivator (rtTA2s–M2) is driven by the 5′ long terminal repeat (5′LTR), and expression of the neomycin resistance gene (neo) is driven by an internal SV40 promoter (SV40). Note that the tet-responsive minimal cytomegalovirus promoter (CMV*−1) is oriented in the opposite orientation to the 5′LTR. cDNAs for BDNF and GFP, respectively, are inserted into the multiple cloning site. Two polyadenylation signals (polyA) are present, one located in the 3′LTR and a bovine growth hormone polyA signal behind the multiple cloning site for the inducible transcript. B, Survival and treatment of animals for histological and ELISA analysis. Duration of treatment with doxycycline in the drinking water is indicated by plus symbols (Dox++++), and survival without doxycycline treatment is indicated by the dashed line (No Dox—-). Note that several groups were first treated with doxycycline followed by doxycycline removal (++++ followed by —-). The number of animals used for histological analysis are indicated at each time point for each treatment group, and numbers in parentheses indicate animal numbers used for BDNF ELISAs.

Article Snippet: BDNF ELISA Ninety-six-well plates were coated with a rabbit anti-BDNF antibody (gift from Amgen, Thousand Oaks, CA) in carbonate buffer using normal rabbit IgG as control.

Techniques: Plasmid Preparation, Retroviral, Expressing, In Vivo, Cloning, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Neuroscience

Article Title: Transient Growth Factor Delivery Sustains Regenerated Axons after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1903-07.2007

Figure Lengend Snippet: Dose dependence and kinetics of BDNF production in primary Fischer 344 rat fibroblasts transfected with tet-on–BDNF. A, With increasing concentrations of doxycycline in the cell culture medium, BDNF expression is increased. B, Kinetics of BDNF expression. tet-on–BDNF transfected cells were cultivated for 3 d with doxycycline (red and black lines) or without doxycycline (blue and green circles). Supernatants were collected beginning at time 0. One group (red line) was changed from doxycycline-containing to doxycycline-free medium, and one group (blue line) was changed from doxycycline-free to doxycycline-containing medium. BDNF levels were measured by ELISA. Cells that were constantly cultivated in doxycycline-free medium (green line) showed very little BDNF expression. Cells that were constantly treated with 1 μg/ml doxycycline (black line) showed continued BDNF expression. Cells that were first in doxycycline-free medium and were then changed to doxycycline-containing culture medium at time 0 (blue line) turned on gene expression within 12 h. This was evidenced by the 24 h supernatant (collected between the 12 and 24 h medium change), which contained high levels of BDNF. Cells that were first cultivated in doxycycline-containing medium and changed to doxycycline-free medium at time 0 (red line) nearly completely shut down gene expression after 24 h. Error bars in A and B are not always visible because of their small size.

Article Snippet: BDNF ELISA Ninety-six-well plates were coated with a rabbit anti-BDNF antibody (gift from Amgen, Thousand Oaks, CA) in carbonate buffer using normal rabbit IgG as control.

Techniques: Transfection, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: The Journal of Neuroscience

Article Title: Transient Growth Factor Delivery Sustains Regenerated Axons after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1903-07.2007

Figure Lengend Snippet: Regulated expression of GFP and BDNF in vivo. A, B, GFP fluorescence in grafts of genetically modified primary fibroblasts in the injured spinal cord 2 weeks after grafting. A, Animals that were treated with doxycycline (+Dox) show GFP fluorescence in the graft, whereas no GFP fluorescence can be found in animals that received GFP grafts in the absence of doxycycline administration (−Dox) (B). Host/graft (g) interface is indicated by dashed lines. C, D, Immunolabeling for GFP shows expression in grafts of animals that were treated with doxycycline for 3 months (C), whereas very weak GFP expression can be found in animals that received GFP grafts in the absence of doxycycline administration (−Dox) for 3 months (D). E, Regulated expression of BDNF in primary fibroblasts grafted to the injured spinal cord. Two weeks after grafting, GFP- and BDNF-expressing grafts were dissected from animals that were either untreated (−Dox) or treated with doxycycline in the drinking water (+Dox) to turn gene expression on. BDNF levels were determined by ELISA. tet-on–BDNF transfected fibroblast grafts from animals that were treated with doxycycline (BDNF + Dox) show significantly higher BDNF levels than untreated animals (BDNF − Dox) and animals with control grafts (***p < 0.001). BDNF levels in untreated animals with tet-on–BDNF transfected fibroblasts grafts are not significantly different from treated (p = 0.76) or untreated (p = 0.85) GFP transfected control animals. Scale bar: A, B, 283 μm; C, D, 138 μm.

Article Snippet: BDNF ELISA Ninety-six-well plates were coated with a rabbit anti-BDNF antibody (gift from Amgen, Thousand Oaks, CA) in carbonate buffer using normal rabbit IgG as control.

Techniques: Expressing, In Vivo, Fluorescence, Genetically Modified, Immunolabeling, Gene Expression, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Cell Proliferation

Article Title: Flavonoid chrysin activates both TrkB and FGFR1 receptors while upregulates their endogenous ligands such as brain derived neurotrophic factor to promote human neurogenesis

doi: 10.1111/cpr.13732

Figure Lengend Snippet: Resource of antibodies.

Article Snippet: Rabbit anti BDNF , ABclonal , A16299.

Techniques: