Journal: Frontiers in Immunology

Article Title: Tissue-Specificity of Antibodies Raised Against TrkB and p75 NTR Receptors; Implications for Platelets as Models of Neurodegenerative Diseases

doi: 10.3389/fimmu.2021.606861

Figure Lengend Snippet: List of antibodies tested.

Article Snippet: p75NTR , Alomone Labs , ANT-007 , Polyclonal , 0.8 µg/ml in 3% BSA , 20 µg/ml.

Techniques: Western Blot, Concentration Assay, Flow Cytometry

Journal: Journal of Clinical Medicine

Article Title: Moderate Aerobic Exercise Regulates Follicular Dysfunction by Initiating Brain-Derived Neurotrophic Factor (BDNF)-Mediated Anti-Apoptotic Signaling Pathways in Polycystic Ovary Syndrome

doi: 10.3390/jcm11195584

Figure Lengend Snippet: Sequences of primers designed for RT-qPCR.

Article Snippet: Sections were incubated overnight at 4 °C with antibodies against BDNF (1:100, Wanleibio), TrkB (1:100, Wanleibio), p75NTR (1:100, 55014-1-AP, Proteintech, Chicago, IL, USA), and Cleaved-Caspase-3 (1:100, 66470-2-Ig, Proteintech) at a 1:100 dilution.

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Moderate Aerobic Exercise Regulates Follicular Dysfunction by Initiating Brain-Derived Neurotrophic Factor (BDNF)-Mediated Anti-Apoptotic Signaling Pathways in Polycystic Ovary Syndrome

doi: 10.3390/jcm11195584

Figure Lengend Snippet: The expression of BDNF, TrkB, and p75NTR in ovarian follicles of PCOS rats after aerobic exercise. Rats received DHEA for the induction of polycystic ovarian syndrome, together with or without exercise treatment. ( A , B ) The expression levels of BDNF in ovarian follicles at different stages were analyzed by immunohistochemistry. ( C , D ) The expression levels of BDNF in ovarian follicles were analyzed by immunofluorescence. ( E , F ) The co-expression of TrkB and p75NTR in ovarian follicles at different stages was detected by immunofluorescence double labeling. n = 10 in each group. Data are shown as mean ± SEM. ** p < 0.05, vs. control group; ## p < 0.01, vs. PCOS group.

Article Snippet: Sections were incubated overnight at 4 °C with antibodies against BDNF (1:100, Wanleibio), TrkB (1:100, Wanleibio), p75NTR (1:100, 55014-1-AP, Proteintech, Chicago, IL, USA), and Cleaved-Caspase-3 (1:100, 66470-2-Ig, Proteintech) at a 1:100 dilution.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Labeling, Control

Journal: Journal of Clinical Medicine

Article Title: Moderate Aerobic Exercise Regulates Follicular Dysfunction by Initiating Brain-Derived Neurotrophic Factor (BDNF)-Mediated Anti-Apoptotic Signaling Pathways in Polycystic Ovary Syndrome

doi: 10.3390/jcm11195584

Figure Lengend Snippet: Aerobic exercise activated BDNF signaling in the ovary of DHEA-induced PCOS rats. Rats received DHEA for the induction of polycystic ovarian syndrome, together with or without exercise treatment. ( A ) mRNA expression of BDNF, TrkB, and p75NTR factors in ovarian tissue was analyzed by real-time PCR. ( B , C ) The expression of BDNF, TrkB, and p75NTR in ovarian tissue was assessed by Western blot assay. ( D , E ) The expression of PI3K, AKT, and p-AKT in ovarian tissue was assessed by Western blot assay. ( F – I ) The expression of p75NTR, NF-κB, p-NF-κB, JNK, and p-JNK in ovarian tissue was assessed by Western blot assay. n = 10 in each group. Data are shown as mean ± SEM. ** p < 0.05, vs. control group; ## p < 0.01, vs. PCOS group.

Article Snippet: Sections were incubated overnight at 4 °C with antibodies against BDNF (1:100, Wanleibio), TrkB (1:100, Wanleibio), p75NTR (1:100, 55014-1-AP, Proteintech, Chicago, IL, USA), and Cleaved-Caspase-3 (1:100, 66470-2-Ig, Proteintech) at a 1:100 dilution.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Frontiers in Molecular Biosciences

Article Title: USF1 Transcriptionally Regulates UGT1A3 and Promotes Lung Adenocarcinoma Progression by Regulating Neurotrophin Signaling Pathway

doi: 10.3389/fmolb.2022.758968

Figure Lengend Snippet: Knockdown of USF1 inhibited cells proliferation, migration and invasion via deregulation of the neurotrophin signaling pathway. (A) The protein expression of p75NTR, RIPK2, IRAK1, IKKβ and USF1, detected by WB in H1299 and A549 cells; (B) The levels of IRAK1 was detected by WB in H1299 and A549 cells; (C,D) Cells migration was detected by wound healing assay in H1299 and A549 cells; (E,F) Cells migration and invasion were detected by transwell assay in H1299 and A549 cells; (The sh-USF1, cells were transfected with sh-USF1; sh-USF1+pcDNA3.1, cells were transfected with sh-USF1 and pcDNA3.1; sh-USF1+ pcDNA3.1- IRAK1; * p < .05 means statistically significant difference when compared with sh-USF1+ pcDNA3.1 group).

Article Snippet: After that, membranes were incubated overnight at 4°C with rabbit polyclonal antibody anti-USF1 (abs115735, 1:500 dilution, Absin, Shanghai, China), rabbit UGT1A3 polyclonal antibody (H00054659-A01, 1:500 dilution, Abnova, Wuhan, China), and rabbit P75 neurotrophic factor receptor (P75NTR) polyclonal antibody (TA328682, 1:200 dilution, OriGene Technologies, Rockville, United States), rabbit receptor interacting serine/threonine kinase 2 (RIPK2) polyclonal antibody (abs130017, 1:500 dilution, Absin, Shanghai, China), rabbit interleukin-1 receptor-associated kinase 1 (IRAK1) polyclonal antibody (AF7290, 1:500 dilution, Beyotime Biotechnology, Shanghai, China), rabbit ikappaB kinase β (IKKβ) polyclonal antibody (AF7200, 1:300 dilution, Beyotime Biotechnology, Shanghai, China), while the rabbit anti-GAPDH polyclonal antibody (1:500, Cell Signaling Technology, Inc.; Shangahi, China) was used as a control.

Techniques: Knockdown, Migration, Expressing, Wound Healing Assay, Transwell Assay, Transfection

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Steady-state distribution and delivery to the plasma membrane of PLAP and NTR-PLAP in transfected FRT cells. (A) Schemes of PLAP and NTR-PLAP (a fusion protein containing the ectodomain of p75NTR and the C-terminal GPI attachment signal of PLAP). (B) FRT cells stably expressing PLAP or NTR-PLAP were grown to confluence and pulsed overnight with [35S]met–cys or [35S]cys, respectively. After surface biotinylation from the apical (Ap) and basolateral (BL) domains, cells were lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (C) FRT cells were labeled for 20 min with [35S]met–cys or [35S]cys. At different times of the chase, surface proteins were biotinylated from the apical (Ap) or the basolateral (BL) side. Cells were then lysed and immunoprecipitated with antibodies against PLAP or NTR and with streptavidin beads. Samples were run on 10% SDS-PAGE and detected by fluorography. PLAP and NTR-PLAP are directly sorted to the apical surface in FRT cells, whereas a small percentage of the two proteins is delivered with similar kinetics to the basolateral domain.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Transfection, Stable Transfection, Expressing, Immunoprecipitation, Labeling, SDS Page

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Pulse-chase analysis and purification on sucrose gradients of transmembrane forms of PLAP and p75NTR in transfected FRT cells. (A) Schemes of p75NTR and PLAP-PS321 (a fusion protein containing the ectodomain of PLAP and the transmembrane and the cytoplasmic domain of the PS321 NTR mutant). (B) Pulse-chase and TX-100 extraction of p75NTR and PLAP-PS321. FRT cells expressing p75NTR and PLAP-PS321 were labeled with [35S]cys or [35S]met–cys, respectively, and subsequently treated as described in Figure ​Figure2A.2A. (C) Purification of p75NTR- and PLAP-PS321–enriched fractions on sucrose density gradients. Labeled proteins of FRT cells expressing p75NTR and PLAP-PS321 were solubilized in TX-100 and purified as described in Figure ​Figure2B.2B. Both transmembrane proteins are soluble in TX-100 and do not float on sucrose density gradients.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Pulse Chase, Purification, Transfection, Mutagenesis, Expressing, Labeling

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Surface expression of GPI-anchored, secretory, and transmembrane proteins after FB1 treatment. (A) FRT cells stably expressing PLAP, NTR-PLAP, or gD1-DAF were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After 30 min of labeling with [35S]met–cys or [35S]cys and a 2-h chase, cells were surface biotinylated from the apical (Ap) or the basolateral (BL) domain, lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (B) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. Apical PLAP and NTR-PLAP, but not basolateral gD1-DAF, became unpolarized after FB1 treatment. (C) FRT cells expressing PLAP-sec, NTR-sec, p75NTR, or PLAP-PS321 were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After labeling with [35S]met–cys or [35S]cys for 20 min and a chase of 2 h, apical and basolateral media were collected separately and cells were surface biotinylated from apical or basolateral domains. Polarized secretion of the secretory forms was revealed by immunoprecipitation in the apical and basolateral medium (top two panels), whereas biotinylated transmembrane proteins were revealed by double immunoprecipitation with specific antibody and streptavidin beads (bottom two panels). (D) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. FB1 does not alter the sorting of transmembrane and secretory proteins.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Expressing, Stable Transfection, Labeling, Immunoprecipitation

Journal:

Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

doi:

Figure Lengend Snippet: Surface expression of GPI-anchored and transmembrane proteins after cholesterol depletion. (A) FRT cells expressing PLAP, NTR-PLAP, gD1-DAF, p75NTR, or PLAP-PS321 were grown on filters and treated with mevinolin (Mev.; 10 μM) for 48 h and M-β-CD (10 mM) for 60 min. Cells were then pulse labeled for 30 min, chased for 2 h, and biotinylated as usual. (B) Amounts of labeled proteins were quantified in three independent experiments and are shown as means ± SD. Hatched bars represent apical (Ap) proteins, and black bars represent basolateral (BL) proteins. Removal of cholesterol does not alter the surface expression of apical and basolateral GPI-anchored and transmembrane proteins.

Article Snippet: The polyclonal antibody against PLAP was from Rockland (Gilbertsville, PA), and anti-p75NTR mAb was a gift from Andrè Le Bivic (Institut de Biologie du Développement de Marseille, Marseille, France).

Techniques: Expressing, Labeling

Journal: Scientific Reports

Article Title: c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

doi: 10.1038/s41598-019-42420-6

Figure Lengend Snippet: Axonal p75 is retrogradely transported in a ligand- and dynein-dependent manner. ( a ) Illustration of the design of the experiment used to study retrograde transport and signaling in sympathetic neurons. ( b ) Kymographs of axonal p75 movement in response to BDNF. Compartmentalized sympathetic neuron cultures were treated with BDNF (200 ng/mL) and MC192-QD, which labeled the extracellular domain of p75, for 4 hours at 37 °C in the axon compartment before imaging. Upper panels show kymographs of retrograde p75 movement in the absence (−) and presence (+) of BDNF in the axon compartment. ( c ) The graph indicates the proportion of vesicles without (black) or with (gray) movement when axon chambers were treated without (−) or with (+) BDNF. Data were obtained from videos captured in three independent experiments. ( d ) Histogram showing the distribution of velocities measured from 105 moving vesicles. The mean velocity of p75 vesicles undergoing retrograde transport in the presence of BDNF was approximately 1.5 µm/second. ( e ) Illustration of the design of the experiment used to study the retrograde transport of p75 and its dependence on dynein. ( f ) Visualization of axonally labeled p75 in the cell bodies of compartmentalized sympathetic neurons. The axons of compartmentalized neuronal cultures were treated with BDNF and MC192-Alexa Fluor 594 (to label p75, red) and microspheres labeled with Alexa Fluor 488 (to label compartmentalized neurons, green) for 16 hours at 37 °C in the absence (control) or presence of the dynein inhibitor EHNA (erythro-9-(2-hydroxy-3-nonyl). Scale bar, 10 μm. ( g ) Levels of axonally labeled p75 in the absence (control) or presence of EHNA. Data from 120 cells in four different compartmentalized chambers were quantified. Microspheres labeled with Alexa Fluor 488 were added 24 hours before the BDNF treatment and maintained in the culture throughout the experiment. Statistically significant differences were analyzed using a two-tailed Mann-Whitney test. ****p < 0,0001.

Article Snippet: NGF, BDNF, anti-BDNF and the monoclonal MC192 anti-p75 antibody were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Labeling, Imaging, Control, Two Tailed Test, MANN-WHITNEY

Journal: Scientific Reports

Article Title: c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

doi: 10.1038/s41598-019-42420-6

Figure Lengend Snippet: Rab5 activity is required for p75 retrograde transport and axonal BDNF-induced death signaling in sympathetic neurons. ( a ) Illustration of the design of the experiment used to study Rab5-dependent p75 retrograde transport and signaling in sympathetic neurons. Quantification ( b ) and visualization ( c ) of axonally labeled p75 in the cell bodies of compartmentalized sympathetic neurons. ( b ) Levels of axonally labeled p75 in the absence (control, GFP) or presence of a dominant negative Rab5 mutant (Rab5DN-GFP). Sixty cells from five different compartmentalized chambers were quantified. Statistically significant differences were analyzed using a two-tailed Mann-Whitney test. ****p < 0,0001. ( c ) After 5 days in culture, sympathetic neurons were transduced with the adenovirus driving the expression of GFP (green) or a dominant negative mutant of Rab5 fused to GFP (Rab5DN-GFP, green). Twenty hours after the infection, the axon compartment was treated with BDNF, MC192-Alexa Fluor 594 (to label p75, red) and the B subunit of the cholera toxin conjugated to Alexa Fluor 647 (B-CTX, cyan) for 16 hours at 37 °C to label compartmentalized neurons. Scale bar, 10 μm. Only cells that were retrogradely labeled with B-CTX-Alexa Fluor 647 were used to quantify the retrograde transport of p75. The white arrow indicates a Rab5DN-GFP labelled neuron with reduced levels of p75. The arrowhead indicates a neuron expressing lower labels of Rab5DN-GFP when compared to the neuron labelled with a white arrow. Consistently, this neuron shows increased levels of p75. The asterisk indicates a neuron that does not express Rab5DN-GFP. This neuron expresses greater levels of p75 compared to neurons labelled with Rab5DN-GFP. ( d ) Illustration of the design of the experiment used to study Rab5-dependent retrograde signaling of p75 and quantify axonal BDNF-induced death signaling in sympathetic neurons. Sixty cells from seven different compartmentalized chambers were quantified at each time point. Statistically significant differences were analyzed using two-way ANOVA and Tukey’s multiple comparison test. ****p < 0,0001. Only cells that were retrogradely labeled with B-CTX-Alexa Fluor 647 were used to quantify apoptosis. ( e-f ) Quantification ( e ) and visualization ( f ) of apoptosis of neuronal cell bodies from compartmentalized sympathetic neuronal cultures expressing GFP and Rab5DN-GFP (in green). Axons were treated with B-CTX-Alexa Fluor 647 (red) to label compartmentalized neurons. Condensed or fragmented nuclei were labeled with Hoechst (blue). The cell enclosed in a white square was magnified to enable the better visualization of the healthy or condensed nucleus labeled with an asterisk. The white arrow indicates a healthy nucleus that is not labeled with B-CTX-Alexa Fluor 647. Scale bar, 10 μm.

Article Snippet: NGF, BDNF, anti-BDNF and the monoclonal MC192 anti-p75 antibody were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Activity Assay, Labeling, Control, Dominant Negative Mutation, Mutagenesis, Two Tailed Test, MANN-WHITNEY, Transduction, Expressing, Infection, Comparison

Journal: Scientific Reports

Article Title: c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

doi: 10.1038/s41598-019-42420-6

Figure Lengend Snippet: JNK activity is required for the retrograde transport and apoptotic signaling triggered by the application of BDNF to the axons of sympathetic neurons. ( a ) Illustration of the design of the experiment used to study the requirement for JNK activation in the retrograde transport of the p75 neurotrophin receptor in SCG neurons. ( b ) and ( c ) Visualization of cell bodies from 6 DIV compartmentalized sympathetic neuron cultures in which the axonal chamber was treated with microspheres-Alexa Fluor 488 (green) and 12.5 mM KCl in the presence of BDNF (200 ng/mL) for 16 hours, and then treated with the vehicles used to dissolve SP600125 or SP600125 ( b ) and or TAT-TI-control peptide or TAT-TI-JIP 153–163 ( c ). Right panels show the quantification of retrogradely transported p75 under control conditions or after treatment with the JNK inhibitors indicated in ( b ) and ( c ). White arrows indicate green microspheres. Data from 200 cells in 7 different compartmentalized chambers were averaged at each time point. Statistical significance was analyzed using a two-tailed Mann-Whitney test. ****p < 0,0001. ( d ) Illustration of the design of the experiment used to study the requirement for JNK activation in death signaling induced by p75 neurotrophin receptor in SCG neurons. ( e ) Visualization of neuronal cell bodies from compartmentalized cultures in which the axonal chamber was treated with BDNF and Alexa Fluor 488-labeled microspheres for 30 hours at 37 °C in the absence (control) or presence of the JNK inhibitor SP600125. Apoptosis was visualized by fragmented nuclei stained with Hoechst (blue), as indicated by white asterisks. Scale bar, 10 μm. ( f ) Percentage of apoptotic cells following the axonal BDNF treatment. Two hundred fifty cells from five different compartmentalized chambers were quantified at each time point. Statistical significance was determined using one-way ANOVA and Tukey’s multiple comparison test. ***p < 0,0002.

Article Snippet: NGF, BDNF, anti-BDNF and the monoclonal MC192 anti-p75 antibody were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Activity Assay, Activation Assay, Control, Two Tailed Test, MANN-WHITNEY, Labeling, Staining, Comparison

Journal: Scientific Reports

Article Title: c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

doi: 10.1038/s41598-019-42420-6

Figure Lengend Snippet: JNK promotes p75 internalization in the axons and cell bodies of sympathetic neurons. ( a ) and ( b ) Visualization of axons of compartmentalized cultures of sympathetic neurons treated with BDNF and MC192-pHRodo in the absence (vehicle) or presence of dynasore for 3–4 hours at 37 °C ( a ), or the absence (vehicle) or presence of SP600125 for 3–4 hours at 37 °C ( b ). The intensity of the pHRodo fluorophore is brighter at an acidic pH, indicating p75 internalization. An inhibitor of p75 internalization, dynasore, reduced the fluorescence associated with p75 internalization. Similar to the effect of dynasore, the presence of SP600125 reduces the fluorescence compared to vehicle conditions. Scale bar, 4,5 µm. Right panels show the quantification of the total fluorescence associated with 366 (vehicle for dynasore), 406 (dynasore), 268 (vehicle for SP600125), 227 (SP600125) axonal segments (9 μm long) from three independent compartmentalized neuronal cultures. Statistically significant differences were analyzed using a two-tailed Mann-Whitney test. ****p < 0,0001. ( c ) and ( d ) Confocal microscopy images of sympathetic neurons treated with BDNF (150 µg/mL) and MC192-Alexa Fluor 594 (3 μg/mL, green, to label p75) for 4 hours at 37 °C in the absence (vehicle and TAT-TI control peptide) or presence of the JNK inhibitors, SP600125 (10 µM) ( c ) or TAT-TI-JIP 153–163 peptide (1 μM) ( d ). Scale bar, 10 μm. Right panels show the levels of internalized p75 after different treatments (relative fluorescence normalized to cell surface p75). Sixty-five cells from three independent experiments were quantified. Statistically significant differences were analyzed using a two-tailed Mann-Whitney test. ****p < 0,0001.

Article Snippet: NGF, BDNF, anti-BDNF and the monoclonal MC192 anti-p75 antibody were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Fluorescence, Two Tailed Test, MANN-WHITNEY, Confocal Microscopy, Control

Journal: Scientific Reports

Article Title: c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

doi: 10.1038/s41598-019-42420-6

Figure Lengend Snippet: JNK is required for the efficient retrograde transport of p75-positive endosomes. ( a ) Schematic of the methodology; the axonal compartment of sympathetic neurons was treated with MC192 conjugated with QD605 and BDNF for 4 hours. Thirty minutes prior to the time-lapse recording, the medium was changed in both compartments to remove the MC192-QDot. Then, the cell body compartment was treated with SP600125 or ciliobrevin D (20 μM) for 30 minutes. Finally, time-lapse images of the cell body compartment were captured at a site proximal to the microgrooves to evaluate the local effects of the inhibitors on the axonal transport of previously endocytosed MC192-QDot. ( b ) Representative kymograph of the retrograde transport of MC192-QDot in axons treated with vehicle, SP600125 or ciliobrevin D. Scale bar, 20 μm. ( c ) Quantification of the number of mobile (>5 μm traveled) and static (<5 μm traveled) particles in axons. Twelve videos from 4 chambers per condition in 2 independent experiments were quantified and analyzed using two-way ANOVA and Tukey’s multiple comparison test. **p < 0.005 and ***p < 0.001. ( d ) Quantification of endosomal flux, as evidenced by the number of dots observed during 30 seconds in 50 μm of axon multiplied by the average velocity of the particles during the time-lapse in each video. Three videos per chamber under each condition were quantified in 2 independent experiments. Student’s t-test: *p < 0.05. ( e ) Graph of the average velocity of the particles under each condition. Fifty-eight to 106 particles per condition were quantified in 2 independent experiment. Statistical significance was determined using Student’s t-test. p = 0.5868. ( f ) Graph indicating the ratio of the average distance traveled by the moving particles under each condition. Fifty-eight to 106 particles per condition were quantified in 2 independent experiments. Statistical significance was determined using Student’s t-test. p = 0.3249. ( g ) In sympathetic neurons axons, BDNF bound to p75 is internalized in a JNK-dependent manner to Rab5 positive endosomes and then is sorted to apoptosis-signaling endosomes for dynein mediated retrograde transport.

Article Snippet: NGF, BDNF, anti-BDNF and the monoclonal MC192 anti-p75 antibody were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Comparison

Journal: Biomedicines

Article Title: A Possible Role for Nerve Growth Factor and Its Receptors in Human Sperm Pathology

doi: 10.3390/biomedicines11123345

Figure Lengend Snippet: Nerve growth factor (NGF) receptors in the ejaculated sperm, epididymis and testis of fertile men. ( A ) The bar graph shows the mean and standard deviation of the fold increase in TrKA (slanted-line bar graph) and p75 NTR (checkerboard bar graph) messenger RNA (mRNA) levels in fertile men ( n = 7). ( B , C ) The plots (blue gate) display the flow cytometric analysis (representative of one fertile man) measuring the size and granularity of sperm ( B ) and their extracellular expression of NGF receptors ( C ). ( D – F ) Fluorescence immunostaining of human sperm from fertile men incubated with the anti-TrKA ( D , E ) and anti-p75 NTR ( F ) antibodies. In ( D , E ), the labelling is evident throughout the entire tail and in the post-acrosomal sheath (( E ), arrow). In ( F ), there is weak staining throughout the entire tail. The nuclei are stained with DAPI, except in ( E ) where the localisation of post-acrosomal sheath is shown. ( G – J ) Immunolocalisation of the anti-TrKA and anti-p75 NTR antibodies in the epididymis ( G and H , respectively) and testis ( I and J , respectively) from a fertile man. Both receptors appear in an apical position of the epididymal epithelium (( G ): TrKA, ( H ): p75 NTR ). There is also clear localisation of both antibodies in the interstitial tissue of the testis (asterisks, ( I ): TrKA, ( J ): p75 NTR ). Bars: 5 µm ( D – F ), 50 µm ( G – J ).

Article Snippet: After washing three times in PBS supplemented with BSA, sperm were incubated at 4 °C for 1 h in PBS/BSA containing FITC-labelled rabbit anti-human p75 NTR extracellular domain (ANT-007-F, Alomone Labs, Jerusalem, Israel) (10 mL/sample) and PE-labelled mouse anti-human TrKA (FAB1751P, R&D Systems, Minneapolis, MN, USA) (10 mL/sample).

Techniques: Standard Deviation, Analysis, Expressing, Fluorescence, Immunostaining, Incubation, Staining, Tissue, FACS

Journal: Biomedicines

Article Title: A Possible Role for Nerve Growth Factor and Its Receptors in Human Sperm Pathology

doi: 10.3390/biomedicines11123345

Figure Lengend Snippet: Nerve growth factor (NGF) receptors in altered sperm from men with infertility. ( A – D ) Fluorescence immunostaining of human sperm from men with infertility (varicocele (V) or urogenital infections (UGIs)) incubated with the anti-TrKA ( A , B ) and anti-p75 NTR ( C , D ) antibodies. In ( A ) (sperm from the UGI group), the TrkA signal is evident in the midpiece of the sperm (arrow); in ( B ) (sperm from V group), there is staining in the midpiece and in the entire coiled tail (asterisk). In ( C , D ), p75 NTR staining is strong in the midpiece and cytoplasmic residue (arrow, sperm from the V group) and in the entire coiled tail (asterisk) and midpiece (( D ), from the UGI group). Bars: 5 µm. ( E , F ) The bar graphs show the mean and standard deviation of the fold change in the TrKA ( E ) and p75 NTR ( F ) mRNA level for fertile men ( n = 7, F, white bars), men with varicocele ( n = 7, V, grey bars) and men with UGI ( n = 7, black bars). One-way analysis of variance followed by the Tukey test was used for multiple comparisons (α = 0.05 and 95% confidence interval). The significant p value (<0.05) is indicated.

Article Snippet: After washing three times in PBS supplemented with BSA, sperm were incubated at 4 °C for 1 h in PBS/BSA containing FITC-labelled rabbit anti-human p75 NTR extracellular domain (ANT-007-F, Alomone Labs, Jerusalem, Israel) (10 mL/sample) and PE-labelled mouse anti-human TrKA (FAB1751P, R&D Systems, Minneapolis, MN, USA) (10 mL/sample).

Techniques: Fluorescence, Immunostaining, Incubation, Staining, Residue, Standard Deviation, Analysis

Journal: Developmental cell

Article Title: Retrograde degenerative signaling mediated by the p75 neurotrophin receptor requires p150 Glued deacetylation by axonal HDAC1

doi: 10.1016/j.devcel.2018.07.001

Figure Lengend Snippet: A. Schematic showing the cleavage of p75NTR

Article Snippet: After 2 days in culture, NGF was removed and the neurons were incubated with a p75NTR antibody (Anti-p75NTR (extracellular)-ATTO-550, Alomone labs) for 30 min in phenol red free DMEM with HEPES, BSA (1 mg/ml) and KCl (12.5 mM) on ice.

Techniques:

Journal: Developmental cell

Article Title: Retrograde degenerative signaling mediated by the p75 neurotrophin receptor requires p150 Glued deacetylation by axonal HDAC1

doi: 10.1016/j.devcel.2018.07.001

Figure Lengend Snippet: A. Inhibition of axonal HDAC1 blocks p75NTR-mediated neuronal apoptosis. Sympathetic neurons were cultured in microfluidic chambers and cell bodies (CB) and distal axons (DAx) were treated with 12.5 mM KCl or 200 ng/ml BDNF +/− 5 μm MS275 (M) for 48 hrs, as indicated. Depicted are the means ± SEM for n=3–4; ***, p < 0.001; ****, p < 0.0001; 2-way ANOVA with a Tukey’s multiple comparisons test.

Article Snippet: After 2 days in culture, NGF was removed and the neurons were incubated with a p75NTR antibody (Anti-p75NTR (extracellular)-ATTO-550, Alomone labs) for 30 min in phenol red free DMEM with HEPES, BSA (1 mg/ml) and KCl (12.5 mM) on ice.

Techniques: Inhibition, Cell Culture

Journal: Developmental cell

Article Title: Retrograde degenerative signaling mediated by the p75 neurotrophin receptor requires p150 Glued deacetylation by axonal HDAC1

doi: 10.1016/j.devcel.2018.07.001

Figure Lengend Snippet: A. Representative images of p75NTR immuno-endocytosis in the presence or absence of BDNF +/− MS275. Sympathetic neurons were incubated with an antibody to p75NTR ECD conjugated to FITC at 4°C then treated for 4 hrs with 12.5 mM KCl or 200 ng/ml BDNF +/− 5 μm MS275 (M) at 37°C, as indicated, then fixed and immunostained for TUJ1 (red). P75NTR is shown in green and nuclei were labeled with DAPI (blue). Cholera toxin B (Ctx B) was used to define the plasma membrane (yellow). Scale bar 10 μm.

Article Snippet: After 2 days in culture, NGF was removed and the neurons were incubated with a p75NTR antibody (Anti-p75NTR (extracellular)-ATTO-550, Alomone labs) for 30 min in phenol red free DMEM with HEPES, BSA (1 mg/ml) and KCl (12.5 mM) on ice.

Techniques: Incubation, Labeling

Journal: Developmental cell

Article Title: Retrograde degenerative signaling mediated by the p75 neurotrophin receptor requires p150 Glued deacetylation by axonal HDAC1

doi: 10.1016/j.devcel.2018.07.001

Figure Lengend Snippet: A. Representative images of an axon from a neuron treated with BDNF. Frames were collected from a time-lapse movie showing p75NTR ICD particles moving in the retrograde (green arrowheads) or anterograde (green arrows) direction or stationary (white arrows). Scale bar:10 μm.

Article Snippet: After 2 days in culture, NGF was removed and the neurons were incubated with a p75NTR antibody (Anti-p75NTR (extracellular)-ATTO-550, Alomone labs) for 30 min in phenol red free DMEM with HEPES, BSA (1 mg/ml) and KCl (12.5 mM) on ice.

Techniques:

Journal: Developmental cell

Article Title: Retrograde degenerative signaling mediated by the p75 neurotrophin receptor requires p150 Glued deacetylation by axonal HDAC1

doi: 10.1016/j.devcel.2018.07.001

Figure Lengend Snippet: A. Inhibition of dynein blocks p75NTR retrograde apoptotic signaling. Sympathetic neurons were cultured in microfluidic chambers and cell bodies (CB) and distal axons (DAx) were treated with 12.5 mM KCl (K) or 200 ng/ml BDNF, as indicated. During the last 8 hrs, 25 μm ciliobrevin A together with BDNF was applied to the distal axons (B+C). The neurons were then fixed and stained for TUJ1 and DAPI to quantify pyknotic nuclei. The bars depict the means ± SEM for n=3; **, p < 0.01, 2-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: After 2 days in culture, NGF was removed and the neurons were incubated with a p75NTR antibody (Anti-p75NTR (extracellular)-ATTO-550, Alomone labs) for 30 min in phenol red free DMEM with HEPES, BSA (1 mg/ml) and KCl (12.5 mM) on ice.

Techniques: Inhibition, Cell Culture, Staining

Journal: Cell reports

Article Title: Macrophage memories of early-life injury drive neonatal nociceptive priming

doi: 10.1016/j.celrep.2024.114129

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: siRNA against p75NTR – Human , Origene , SR321107.

Techniques: Recombinant