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: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Induction of differentiation using the Ex.1 method. (A) Colony of human iris-derived iPS cells. (B-D) Cell colony in differentiation culture: (B) 6th day in STEP-1 medium, (C) 18th day in STEP-2 medium, and (D) 25th day in STEP-3 medium. Scale bar in (A-D), 100 µm. (E) Fold-increase in p75NTR mRNA expression level at different steps relative to STEP-0: * P<0.05. (F) Cell morphology on 35th day of differentiation in STEP-3 medium. (G) Immunostaining for αA-crystallin in the same area as indicated in (F). (H) Immunostaining for SOX2 in the same area as in (G). (I) Merged image of αA-crystallin and SOX2 immunostaining. Scale bar in (F-I), 100 µm. Ex, experiment; iPS cells, induced pluripotent stem cells.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Derivative Assay, Expressing, Immunostaining

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Changes in gene expression levels at each step of differentiation induction using Ex.4 the method. (A) SOX2 , (B) PAX6 , (C) p75NTR , (D) αA-crystallin , (E) type-IV collagen ; fold-changes are relative to gene expression level in STEP-0: * P<0.05, ** P<0.01, *** P<0.005. Ex, experiment.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Formation of three-dimensional cell aggregates expressing lens-specific proteins in various cultures of human iris-derived tissue cells and iPS cells

doi: 10.3892/etm.2022.11476

Figure Lengend Snippet: Induction of differentiation using the Ex.4 method. (A) Gross observation of cell aggregate. Reduced opacity is observed in certain areas. Scale bar, 2 mm. (B) H&E-stained specimen of cell aggregate. Scale bar, 200 µm. (C) Enlarged view of H&E staining in Area 1 of the specimen in (B). Cells displaying epithelial-like morphology have infiltrated inside the cell aggregate (yellow arrowhead). (D-G and I-L) Immunostaining of cell aggregates for (D and I) SOX2, (E and J) p75NTR, (F and K) αA-crystallin, and (G and L) type IV collagen. Blue in immunostaining images: DAPI staining of nuclei. (H) Enlarged view of H&E staining of Area 2 in the specimen in (B), where the cells on the surface of the aggregate have transitioned from a multilayer to a monolayer (white arrowhead). Scale bar in (C-L), 50 µm. Ex, experiment; H&E, hematoxylin and eosin.

Article Snippet: Briefly, fixed cells were permeabilized with 0.5% Triton X-100 (cat. no. 04605-250; FUJIFILM Wako), blocked with a serum-free ready-to-use blocking reagent (cat. no. X090930-2; Agilent Technologies, Inc.) for 5 min at room temperature, and stained with one of the following primary antibodies (incubated for 1 h at 37˚C): anti-human αA-crystallin rabbit polyclonal antibody (1:100; cat. no. ab5595; Abcam plc.), anti-human PAX6 mouse monoclonal antibody (1:100; cat. no. 14-9914-80; Thermo Fisher Scientific), anti-human SOX2 rat monoclonal antibody (1:100; cat. no. 14-9811-82; Thermo Fisher Scientific), anti-human type IV collagen rabbit polyclonal antibody (1:200; cat. no. LB-0445; Life Science Laboratories, Inc.), anti-p75NTR rabbit polyclonal antibody (1:200; cat. no. ANT-007; Alomone Labs), and anti-human βB2-crystallin rabbit polyclonal antibody (1:100; cat. no. ab252971; Abcam).

Techniques: Staining, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

doi: 10.3390/ijms23020892

Figure Lengend Snippet: BoNT/A blocked NGF-induced CGRP release and -enhancement of 20 nM CAP-evoked CGRP release. ( A ) After 2 days in the presence of 50 ng/mL NGF, TGNs were starved of the neurotrophin as detailed before, without or with the inclusion of 100 nM BoNT/A during this latter step. The release experiment was performed as described in A. ( B ) At the end of the protocol, one well each of BoNT/A-treated and non-treated cells were solubilised in 1× LDS and subjected to Western blotting. PVDF membranes were cut horizontally midway between the 25 k and 37 k molecular weight markers. The upper portion was exposed to antibodies reactive with syntaxin-1 (mouse monoclonal, 1:2000) and the lower piece probed with an antibody recognising both intact and BoNT/A-truncated SNAP-25 (mouse monoclonal, 1:3000). ( C ) The amount of cleaved SNAP-25 in BoNT/A-treated cells was calculated as a % (mean + s.e.m., N = 3) of the total SNAP-25 (intact + BoNT/A product). ( D ) Total CGRP (pg/well). ( E – G ) Histograms showing: ( E ) spontaneous CGRP release during the second 30 min. incubation into HBS without (grey bars) or induced by 100 ng/mL NGF (blue bars), ( F ) during the third period evoked by 20 nM CAP (minus the spontaneous release), and ( G ) during the third incubation with 1 µM CAP minus the spontaneous release. In all cases, CGRP release is expressed as a % of total CGRP (mean + s.e.m., N = 3, n = 9). Asterisks summarise the results of unpaired one-tailed Welch tests applied to the data plotted in panels E, F, and G, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: NGF 2.5S and antibodies to NGF (AN-240) and TRPV1 (ACC-030; used to determine TRPV1 expression by Western blotting) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Western Blot, Molecular Weight, Incubation, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

doi: 10.3390/ijms23020892

Figure Lengend Snippet: Chimera/EA effectively inhibits 1 µM CAP-evoked CGRP release from starved TGNs, and its enhancement by NGF. TGNs were starved and incubated with 100 nM/EA using a protocol identical to that described previously for BoNT/A ( A). ( A ) Western blotting with anti-SNAP-25 antibodies confirms the disappearance of intact SNAP-25 and the appearance of a much faster-migrating product in cells exposed to/EA (+) but not control (−). ( B ) Histogram displaying the % of SNAP-25 cleaved, which was calculated as in C. ( C ) Total amounts (pg/well) of CGRP, determined as before, in control and/EA-treated cells. ( D ) Amounts of spontaneous CGRP release (% total) into HBS only, (grey bars), and that during incubation with 100 ng/mL NGF (blue bars), and ( E ) upon stimulation with 1 µM CAP (minus the spontaneous release) (mean + s.e.m. N = 3, n = 9). Unpaired one-tailed Welch test was applied to the data plotted in panels ( C – E ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: NGF 2.5S and antibodies to NGF (AN-240) and TRPV1 (ACC-030; used to determine TRPV1 expression by Western blotting) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Incubation, Western Blot, Control, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases

doi: 10.3390/ijms23020892

Figure Lengend Snippet: NGF induces a minor increase in Ca 2+ - and SNARE-dependent CGRP release, whereas it greatly enhances the CAP-evoked exocytosis which is blocked by BoNT/A and/EA at low [CAP] but only abolished by BoNT/EA at higher [CAP]. ( A ) Illustrates the effect of acute NGF on CGRP exocytosis from control neonatal rat TGNs starved of the neurotrophin for 2 days, and ( B ) in TGNs pre-treated with BoNT/A or /EA. ( A ) NGF binds to its receptor TrkA, activates the signalling cascades shown , and induces Ca 2+ influx by an unidentified mechanism ( ? ). Elevated intracellular Ca 2+ ([Ca 2+ ] i ) triggers the fusion of large dense core vesicles (LDCVs) via SNARE-complexes (VAMP, syntaxin-1 and SNAP-25), thereby, causing exocytotic release of CGRP and surface delivery of vesicle constituents. This acute potentiation by NGF can involve the phosphatidylinositol 3-kinase—Src (PI3K-Src) pathway, which promotes trafficking of LDCVs, and insertion of their TRPV1 channels into the plasmalemma by Ca 2+ -regulated exocytosis (blue arrows) c.f. [ , ]. Additionally, the phospholipase C γ (PLCγ) cascade leads to sensitisation of TRPV1 already on the plasmalemma (red dashed arrows) . The outcome of these composite influences of NGF on TRPV1 is that when the channel is activated by CAP [Ca 2+ ] I is raised even more than normally [ , , ] and this further enhances CGRP release . ( B ) The proteases of BoNT/A and/EA delete 9 (purple arrow) and 26 (green arrow) residues from SNAP-25 (Insert), respectively, preventing the fusion of LDCVs; this blocks the minimal CGRP exocytosis elicited by NGF (arrow with crosses, left) and its enhancement of the release evoked by 20 nM CAP ( ) (arrow with crosses, centre). Stronger stimulation of TRPV1 with 1 µM CAP ( ) induces a lot more Ca 2+ influx ( [Ca 2+ ] i , ( [Ca 2+ ] i , ( [Ca 2+ ] i ; ) which causes a moderate increase in CGRP release but overcomes the inhibition by BoNT/A (purple cross with broken lines, right) while/EA (green cross, right) remains effective in diminishing CGRP release. Acute sensitisation by NGF of TRPV1 selectively enhances neuropeptide exocytosis stimulated by low [CAP] (<100 nM) and only moderately affects responses to ≥100 nM CAP ( B). Despite being impotent against 1 µM CAP-evoked CGRP release in starved cells (( B ), , and G), BoNT/A partially inhibits the moderate NGF-enhancement of 1 µM CAP-evoked CGRP release ( G), implicating membrane trafficking in the sensitisation process (B, PI3K-Src stimulated pathway) in accordance with its inhibition of NGF-induced, Ca 2+ -dependent CGRP exocytosis ( E).

Article Snippet: NGF 2.5S and antibodies to NGF (AN-240) and TRPV1 (ACC-030; used to determine TRPV1 expression by Western blotting) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Control, Inhibition, Membrane

Journal: European journal of pharmacology

Article Title: Fabry disease-associated globotriaosylceramide induces mechanical allodynia via activation of signaling through proNGF-p75 NTR but not mature NGF-TrkA.

doi: 10.1016/j.ejphar.2021.173882

Figure Lengend Snippet: Fig. 2. Signaling through proNGF–p75NTR, but not mNGF–TrkA, is required for Gb3- induced mechanical sensitization. (A) Experimental timeline of the von Frey tests and the i.pl. injection of Gb3 and anti body/antiserum or antibody/antiserum alone. (B, C) The antiserum (1 μl/paw, i.pl.) containing anti-proNGF antibody effectively recovered the Gb3-induced decrease of the 50% paw withdrawal threshold (PWT; 1 h: H = 9.40, P = 0.009; 3 h: H = 9.13, P = 0.010; 6 h: H = 6.79, P = 0.034; 24 h: H = 6.24, P = 0.044) and its area under the curve (AUC; H = 10.1, P = 0.006). n = 8 per group. The injection volume was 10 μl. (D, E) The anti-NGF antibody (1 μg/paw and 10 μg/ paw, i.pl.) failed to significantly alleviate reductions in the pain threshold and its AUC (H = 10.5, P = 0.015) in Gb3-injected mice. Vehicle, n = 7; Gb3 + Control IgG, n = 8; Gb3 + Anti-NGF antibody (1 μg), n = 9; Gb3 + Anti-NGF antibody (10 μg), n = 6. The injection volume was 10 or 20 μl. (F, G) Administration of an anti-p75NTR antibody (1 μg/paw, i.pl.) almost completely reversed the Gb3-induced decline of the 50% PWT (24 h: H = 8.44, P = 0.015) and its AUC (H = 9.16, P = 0.010) to the level of the vehicle control. Vehicle, n = 11; Gb3 + Control IgG, n = 15; Gb3 + Anti-p75NTR antibody, n = 14. The injection volume was 10 or 20 μl. (H, I) Inhibition of TrkA activity with the neutral izing antibody (1 μg/paw and 10 μg/paw, i. pl.) gave minimal influence on the Gb3- induced mechanical allodynia (AUC: H = 9.80, P = 0.020). Vehicle, n = 11; Gb3 + Control IgG, n = 11; Gb3 + Anti-TrkA anti body (1 μg), n = 7; Gb3 + Anti-TrkA anti body (10 μg), n = 7. The injection volume was 20 μl. *P < 0.05, **P < 0.01 vs the Vehicle group, #P < 0.05, ##P < 0.01 vs the Gb3 + Normal serum or Control IgG group by Dunn’s test with Holm adjustment.

Article Snippet: The details of the NGF-related antibodies/antiserum and their control IgG/serum were as follows: mouse monoclonal anti-NGF antibody (L148M, Exalpha Biologicals, Shirley, MA, USA; 1 μg and 10 μg, Shutov et al., 2016); sheep polyclonal anti-proNGF antibody (S-080-100, Biosensis, Thebarton, Australia; 1 μl J. Sugimoto et al. European Journal of Pharmacology 895 (2021) 173882 antiserum); mouse monoclonal anti-TrkA antibody (Ab00439-1.4, Absolute Antibody, Redcar, UK; 1 μg and 10 μg, Ugolini et al., 2007); rabbit polyclonal anti-p75NTR antibody (AB-N01AP, Advanced Targeting Systems, San Diego, CA, USA; 1 μg, Watanabe et al., 2008); normal mouse IgG as a control of anti-NGF or anti-TrkA antibody (0G11, Exalpha Biologicals; Ab00178-1.4, Absolute Antibody); normal rabbit IgG as a control of anti-p75NTR antibody (PM035, MBL, Nagoya, Japan); and normal sheep serum as a control of anti-proNGF antibody (S3772, Sigma-Aldrich).

Techniques: Injection, Control, Inhibition, Activity Assay

Journal: European journal of pharmacology

Article Title: Fabry disease-associated globotriaosylceramide induces mechanical allodynia via activation of signaling through proNGF-p75 NTR but not mature NGF-TrkA.

doi: 10.1016/j.ejphar.2021.173882

Figure Lengend Snippet: Fig. 3. Gb3 injection did not lead to elevation of proNGF and mNGF or their receptor expression in either plantar skin or dorsal root ganglia. There were no changes in proNGF (A, C) nor mNGF (B, D) content in the plantar skin (A, B) and dorsal root ganglia (DRG; C, D) in mice that were 1 h or 24 h post-Gb3 treatment. n = 9 per group. The injection volume was 10 or 20 μl. Gb3 injection did not modify the expression levels of p75NTR in the plantar skin (E, 1 h: n = 6 per group; 24 h: n = 7 per group) or the DRG (F, n = 7 per group). The injection volume was 10 μl. The Vehicle (Veh) group, open circles; the Gb3 group, closed circles; MW, molecular weight.

Article Snippet: The details of the NGF-related antibodies/antiserum and their control IgG/serum were as follows: mouse monoclonal anti-NGF antibody (L148M, Exalpha Biologicals, Shirley, MA, USA; 1 μg and 10 μg, Shutov et al., 2016); sheep polyclonal anti-proNGF antibody (S-080-100, Biosensis, Thebarton, Australia; 1 μl J. Sugimoto et al. European Journal of Pharmacology 895 (2021) 173882 antiserum); mouse monoclonal anti-TrkA antibody (Ab00439-1.4, Absolute Antibody, Redcar, UK; 1 μg and 10 μg, Ugolini et al., 2007); rabbit polyclonal anti-p75NTR antibody (AB-N01AP, Advanced Targeting Systems, San Diego, CA, USA; 1 μg, Watanabe et al., 2008); normal mouse IgG as a control of anti-NGF or anti-TrkA antibody (0G11, Exalpha Biologicals; Ab00178-1.4, Absolute Antibody); normal rabbit IgG as a control of anti-p75NTR antibody (PM035, MBL, Nagoya, Japan); and normal sheep serum as a control of anti-proNGF antibody (S3772, Sigma-Aldrich).

Techniques: Injection, Expressing, Molecular Weight

Journal: Veterinary pathology

Article Title: p75 Neurotrophin Receptor: A Double-Edged Sword in Pathology and Regeneration of the Central Nervous System.

doi: 10.1177/0300985818781930

Figure Lengend Snippet: Figure 1. Schematic representation of the various interactors of nerve growth factor receptor p75 (p75NTR). The extracellular domain binds neurotrophins as well as their respective proforms (red). Inter- action with other receptors (gray) influences the outcome of p75NTR

Article Snippet: Furthermore, the following antibodies have been tested for immunohistochemistry in canine and murine samples and specifically reacted with p75NTR in our lab: polyclonal p75NTR antibody (extracellular domain; Novus Biologicals NBP1-25971) and polyclonal p75NTR antibody (intracellular domain; Alomone labs ANT-011).

Techniques:

Journal: Veterinary pathology

Article Title: p75 Neurotrophin Receptor: A Double-Edged Sword in Pathology and Regeneration of the Central Nervous System.

doi: 10.1177/0300985818781930

Figure Lengend Snippet: Figure 14. Antigen expression in different Schwann cell stages (sche- matic representation). Markers in the violet box are expressed in immature and nonmyelinating Schwann cells but not in myelinating Schwann cells. GalC (blue) is demonstrated in both myelinating and nonmyelinating Schwann cells. a1b1 integrin (yellow) and markers in the green box are specific for nonmyelinating and myelinating Schwann cells, respectively. GalC, galactocerebrosid; GAP-43, growth- associated protein 43; GFAP, glial fibrillary acidic protein; Krox-20, early growth response protein 2 (EGR2); MAG, myelin-associated protein; MAL, myelin and lymphocyte protein; MBP, myelin basic pro- tein; NCAM, neural cell adhesion molecule; p75NTR, nerve growth factor receptor; PLP, proteolipid protein; PMP-22, peripheral myelin protein 22; P2, peripheral myelin protein 2 (PMP2); Ran-2, rat neural antigen-2. Modified from Mirsky et al and Lempp et al.121,135

Article Snippet: Furthermore, the following antibodies have been tested for immunohistochemistry in canine and murine samples and specifically reacted with p75NTR in our lab: polyclonal p75NTR antibody (extracellular domain; Novus Biologicals NBP1-25971) and polyclonal p75NTR antibody (intracellular domain; Alomone labs ANT-011).

Techniques: Expressing, Modification

Journal: bioRxiv

Article Title: p57Kip2 regulates embryonic haematopoietic stem cell numbers by controlling the size of the sympathoadrenal progenitor pool

doi: 10.1101/2021.09.13.459835

Figure Lengend Snippet: Immunohistochemistry on cryosections from E11 wild-type embryos. ( A ) Immunostaining for p57Kip2 (red) and CD34 (green) with DAPI nuclear stain (blue). P57Kip2 expression is highlighted in endothelial cells (arrowheads), sub-endothelial mesenchyme (asterisk) and SA cells (arrows). ( B ) Immunostaining for p57Kip2 (red) and Ngfr (green) with DAPI nuclear stain (blue). P57Kip2 expression is highlighted in sub-endothelial mesenchyme (asterisk) and SA cells (arrows). Dashed line shows outline of the aorta. ( C ) Immunostaining for Pdgfrβ (red) with DAPI nuclear stain (blue). Exclusion of Pdgfrβ expression from SA cells is highlighted (arrows). Scale bars indicate 50μm. ( D ) Sorting strategy for AGM subpopulations; HSPCs, CD34+CD45+; endothelial cells (EC), CD34+CD45-; SA cells, Ngfr+Pdgfrβ-; mesenchymal cells (ME), Ngfr-Pdgfrβ+. Gating was based on fluorescence minus one controls (FMOs) as shown in Figure S1. ( E, F ) p57Kip2 mRNA expression (relative to β actin ) by qPCR in subpopulations sorted from E11 and E12 AGMs. n ≥ 3. Histogram represents mean ±SEM.

Article Snippet: The following antibodies were used: CD45.1 (A20), CD45.2 (104), CD34 (RAM34), Pdgfrβ (APB5), CD45 (104), Ngfr (polyclonal; ANT-007-AO; Alomone Labs Ltd).

Techniques: Immunohistochemistry, Immunostaining, Staining, Expressing, Fluorescence

Journal: bioRxiv

Article Title: p57Kip2 regulates embryonic haematopoietic stem cell numbers by controlling the size of the sympathoadrenal progenitor pool

doi: 10.1101/2021.09.13.459835

Figure Lengend Snippet: ( A ) Immunohistochemical staining of E11 wild-type embryo cryosection for Ngfr (green), p57Kip2 (red) and Th (white). Bar represents ventral vs dorsal subdissection. ( B ) t-SNE plots coloured for clusters identified by k-means clustering (left) and for ventral and dorsal origin (right). Violin plots for expression levels of Ngfr ( C ) and haematopoiesis-associated genes Ptprc / CD45 ( D ), Csf1r ( E ), Cx3cr1 ( F ), Runx1 ( G ), Procr / EPCR ( H ), Kit ( I ) and Cd34 ( J ). ( K ) t-SNE coloured for predicted cell cycle phases (top) and the identified clusters before (middle) and after (bottom) cell cycle correction. ( L ) t-SNE plot of remaining 4 clusters following cell cycle correction (bottom) and coloured according to ventral and dorsal origin (top). t-SNE gene expression plots coloured for expression levels of SNS genes Sox10 ( M ), Th ( N ), Phox2a ( O ), Gata3 ( P ), Phox2b ( Q ) and Cdkn1c/p57Kip2 ( R ).

Article Snippet: The following antibodies were used: CD45.1 (A20), CD45.2 (104), CD34 (RAM34), Pdgfrβ (APB5), CD45 (104), Ngfr (polyclonal; ANT-007-AO; Alomone Labs Ltd).

Techniques: Immunohistochemical staining, Staining, Expressing, Gene Expression

Journal: JOR Spine

Article Title: Age‐related molecular changes in the lumbar dorsal root ganglia of mice: Signs of sensitization, and inflammatory response

doi: 10.1002/jsp2.1124

Figure Lengend Snippet: List of primary and secondary antibodies used for immunofluorescence analysis

Article Snippet: rabbit anti‐NGF , BosterBio , m00341 , 1.00 , 1:100 , same as above.

Techniques: Immunofluorescence, Concentration Assay