Journal: International Journal of Molecular Sciences

Article Title: The Slow-Releasing and Mitochondria-Targeted Hydrogen Sulfide (H 2 S) Delivery Molecule AP39 Induces Brain Tolerance to Ischemia

doi: 10.3390/ijms22157816

Figure Lengend Snippet: The list of primary and secondary antibodies used in Western blot and fluorescence microscopy techniques.

Article Snippet: pro-NGF , Alomone , AGP-031 , 1:300.

Techniques: Western Blot, Fluorescence, Microscopy, Marker

Journal: FEBS Open Bio

Article Title: ProNGF processing in adult rat tissues and bioactivity of NGF prodomain peptides

doi: 10.1002/2211-5463.13768

Figure Lengend Snippet: (A–C) Dot blot and western blot for commercial antibody selectivity towards proNGF and/or NGF. (A) Detection of NGF, proNGF, C1 and C6 by using the different antibodies against NGF and proNGF: Abcam ab6199, Alomone ANT‐005 and Biosensis M‐1778‐B or S‐080. (B, C) The representative western blots show the ability and selectivity of (B) Abcam anti‐NGF antibody and (C) Biosensis (M‐1778‐B) anti‐proNGF antibody to detect recombinant NGF and/or proNGF.

Article Snippet: To detect NGF and proNGF, Abcam antibody (ab6199) against NGF and anti‐proNGF antibodies from Biosensis (M‐1778‐B and S‐080) and Alomone (ANT‐005) were used.

Techniques: Dot Blot, Western Blot, Recombinant

Journal: Frontiers in Immunology

Article Title: Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis

doi: 10.3389/fimmu.2022.818630

Figure Lengend Snippet: proNGF increases the expression of inflammatory cytokines in synovial fibroblasts from RA patients. (A) The addition of exogenous proNGF (200ng/ml) together with IL-1β at suboptimal doses (10pg/ml and 50pg/ml) induces a synergic increase in IL-6 production not observed at higher concentration of IL-1β. The experiments (n = 4) were performed in serum free medium. Data were analyzed by paired t -test (*p < 0.05). (B–D) Inhibition of proNGF binding to p75NTR with LM11A-31 (10nM) decreases inflammatory mediators: IL-6 (B) , IL-8 (C) and MCP1 (D) production was significantly reduced in RA-FLS cells activated using 100 ng/ml TNF-α, 100 ng/ml LPS or 1 ng/ml IL-1β. RA-FLS were cultured for 18 hours in 10% FBS DMEM. The inhibitory effect of LM11A-31 on cytokine release is expressed as percentage decrease (% decrease) from activated cells. (E) To obtain a confirm of the effects of p75NTR blocking, p75NTR was neutralized using a specific anti-p75NTR antibody (2,5 µg/ml) and the release of IL-6 was measured in RA-FLS activated with 100 ng/ml TNF-α, 100 ng/ml LPS or 1ng/ml IL-1β. In this set of experiments (n = 6) cells were cultured in 10% FBS DMEM for 18 hours. The results are expressed as percentage decrease (% decrease) from activated cells.

Article Snippet: RA-FLS and OA-FLS were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 PBS, incubated for 1 hour at RT with 1% BSA, 5% goat serum (Abcam, Cambridge, UK) PBS and then with mouse anti-p75NTR antibody (Merck, Darmstadt, Germany, clone 8211) or rabbit anti-proNGF (Merck AB9040) and Alexa Fluor secondary antibodies (Invitrogen).

Techniques: Expressing, Concentration Assay, Inhibition, Binding Assay, Cell Culture, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis

doi: 10.3389/fimmu.2022.818630

Figure Lengend Snippet: proNGF present in synovial fluids increases the expression of inflammatory mediators in synovial fibroblasts from RA patients. (A) proNGF is by far the most abundant NGF form (100 to 200-fold that of mature NGF) detected in synovial fluids (SF) obtained from six active patients (1-6). (B) To recreate ex vivo inflamed synovia condition, 30% v/v synovial fluid was added to RA-FLS (n = 19) cultured in 10% FBS DMEM. Data were analyzed by paired t -test (***p < 0.001). (C) RA-FLS in 30% v/v synovial fluid were treated with 10 nM of LM11A-31 with LM11A-31 and IL-6 production was measured after 18 hours. The data represent the percentage of inhibition obtained in 19 independent experiments performed using seven different RA-FLS and synovial fluids obtained from different patients (n = 18). Data were analyzed by one sample t -test (***p < 0.001). (D) 30% v/v synovial fluid was added to RA-FLS cultured in 10% FBS DMEM with or without the addition of anti-IL1β (5µg/ml) (n = 9). IL-6 release was measured after 18 hours of incubation. Data were analyzed by one sample t-test (*p < 0.05). (E) The apoptosis rate of RA-FLS treated with 30% v/v of synovial fluids with or without the addition of LM11A-31 at two different doses (10nM used for all our experiment and a ten-fold higher dose 100 nM) was analyzed by Annexin V/PI staining. No modification in the percentage of apoptotic cells was observed following the addition of synovial fluid or after p75NTR inhibition with LM11A-3 with both the doses used. Representative scatterplots of three independent experiments are shown.

Article Snippet: RA-FLS and OA-FLS were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 PBS, incubated for 1 hour at RT with 1% BSA, 5% goat serum (Abcam, Cambridge, UK) PBS and then with mouse anti-p75NTR antibody (Merck, Darmstadt, Germany, clone 8211) or rabbit anti-proNGF (Merck AB9040) and Alexa Fluor secondary antibodies (Invitrogen).

Techniques: Expressing, Ex Vivo, Cell Culture, Inhibition, Incubation, Staining, Modification

Journal: Frontiers in Immunology

Article Title: Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis

doi: 10.3389/fimmu.2022.818630

Figure Lengend Snippet: The pro-inflammatory p75NTR/proNGF loop in RA-FLS. (A) Inflammatory stimuli activate an autocrine loop involving proNGF and p75NTR. Inflammatory stimuli strongly induce the contemporary expression of p75NTR, the specific proNGF receptor, and of its ligand, proNGF, in RA-FLS. The increased p75NTR expression results in a higher binding capacity of RA-FLS for proNGF, whose endogenous production is strongly induced by inflammatory stimuli. (B) The active p75NTR/proNGF axis enhances inflammatory cytokines production. The high amounts of endogenously-produced proNGF, released by activated RA-FLS, interact with the p75NTR receptors highly expressed on RA-FLS membrane. The activation of p75NTR intracellular pathways (i.e. p38 and JNK) results in an amplified production of inflammatory cytokines (i.e. IL-6). (C) Inhibition of the p75NTR/proNGF loop decreases inflammatory cytokines production. The blocking of p75NTR using LM11A-31, a small molecule that specifically blocks the binding site of p75NTR for proNGF, results in a net reduction of inflammatory cytokine production (i.e. IL-6). The endogenous proNGF released by active RA-FLS cannot bind to p75NTR and, consequently, does not activate p75NTR intracellular pathways that induce inflammatory cytokine production.

Article Snippet: RA-FLS and OA-FLS were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 PBS, incubated for 1 hour at RT with 1% BSA, 5% goat serum (Abcam, Cambridge, UK) PBS and then with mouse anti-p75NTR antibody (Merck, Darmstadt, Germany, clone 8211) or rabbit anti-proNGF (Merck AB9040) and Alexa Fluor secondary antibodies (Invitrogen).

Techniques: Expressing, Binding Assay, Produced, Activation Assay, Amplification, Inhibition, Blocking Assay

Journal: Oncotarget

Article Title: ProNGF is a potential diagnostic biomarker for thyroid cancer

doi: 10.18632/oncotarget.8652

Figure Lengend Snippet: A–D. Immunohistochemical detection of proNGF was performed with a polyclonal antibody on a series of thyroid cancers (n=40), adenoma (n=40) and normal thyroid tissues (n=80). ProNGF was found in epithelial cells with a marked increased in cancer tissues. Representative pictures are shown for normal thyroid tissue (A), adenoma (B), papillary carcinoma (C), follicular carcinoma (D). Scale = 50μm. E. Quantification of proNGF staining intensities was performed using the Halo™ image analysis platform, h-scores were calculated and used to establish the ROC curves. ProNGF staining intensities were significantly higher for cancers (median h-score = 69.3) than adenomas (median h-score = 35.5) and normal tissues (median h-score = 19.7) (p<0.0001). The box limits indicate the 25th and 75th percentiles with the whiskers extending 1.5 times the interquartile range from the 25th and 75th percentiles (outliers are represented by dots) (****p<0.0001). F–H. ROC curves for proNGF staining intensity levels in thyroid cancers versus adenomas and normal thyroid tissues were established and analyzed using GraphPad™. The area under the curve was 0.99 (95% CI 0.98-1.00, p<0.0001) for cancers versus normal samples (F), 0.84 (95% CI 0.75-0.93, p<0.0001) for cancers versus adenomas (G), and 0.95 (95% CI 0.85-0.96, p<0.0001) for cancers versus adenomas and normal samples (H).

Article Snippet: A polyclonal anti-proNGF antibody (Merck Millipore, Darmstadt, Germany, catalogue number AB9040) was used at 1/200 for cohort 1, and a monoclonal anti-proNGF antibody made against the proNGF propeptide sequence (in house from Biomerieux, Marcy l'Etoile, France) was used at 2.5 μg/ml for cohort 2.

Techniques: Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: ProNGF is a potential diagnostic biomarker for thyroid cancer

doi: 10.18632/oncotarget.8652

Figure Lengend Snippet: ProNGF expression in thyroid cancers versus adenomas and normal tissues (cohort 1) and associations with clinicopathological parameters

Article Snippet: A polyclonal anti-proNGF antibody (Merck Millipore, Darmstadt, Germany, catalogue number AB9040) was used at 1/200 for cohort 1, and a monoclonal anti-proNGF antibody made against the proNGF propeptide sequence (in house from Biomerieux, Marcy l'Etoile, France) was used at 2.5 μg/ml for cohort 2.

Techniques: Expressing

Journal: Oncotarget

Article Title: ProNGF is a potential diagnostic biomarker for thyroid cancer

doi: 10.18632/oncotarget.8652

Figure Lengend Snippet: A-F. Immunohistochemical detection of proNGF in thyroid cancers of various histological types (n=127 cases), adenomas (n=6) and normal thyroid tissues (n=55) was performed witha monoclonal antibody. ProNGF was found in epithelial cells with a marked increased in cancer tissues. Representative pictures are shown for normal thyroid tissue (A), adenoma (B), papillary carcinoma (C), follicular carcinoma (D), medullary carcinoma (E), and anaplastic carcinoma (F). Scale = 50μm. G. Quantification of proNGF staining intensities was performed using the Halo™ image analysis platform, h-scores were calculated and used to establish the ROC curves. ProNGF staining intensities were significantly higher for cancers (median h-score = 54.6) than adenomas (median h-score = 21.7) and normal tissues (median h-score = 7.2). The box limits indicate the 25th and 75th percentiles with the whiskers extending 1.5 times the interquartile range from the 25th and 75th percentiles (outliers are represented by dots) (****p<0.0001). H. The ROC curve for proNGF staining intensity levels in thyroid cancers versus normal thyroid tissues was established and analyzed using GraphPad™. The area under the ROC curve was 0.98 (95% CI 0.97-0.99, p<0.0001).

Article Snippet: A polyclonal anti-proNGF antibody (Merck Millipore, Darmstadt, Germany, catalogue number AB9040) was used at 1/200 for cohort 1, and a monoclonal anti-proNGF antibody made against the proNGF propeptide sequence (in house from Biomerieux, Marcy l'Etoile, France) was used at 2.5 μg/ml for cohort 2.

Techniques: Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: ProNGF is a potential diagnostic biomarker for thyroid cancer

doi: 10.18632/oncotarget.8652

Figure Lengend Snippet: ProNGF expression in thyroid cancers of different histological types (cohort 2) and associations with clinicopathological parameters

Article Snippet: A polyclonal anti-proNGF antibody (Merck Millipore, Darmstadt, Germany, catalogue number AB9040) was used at 1/200 for cohort 1, and a monoclonal anti-proNGF antibody made against the proNGF propeptide sequence (in house from Biomerieux, Marcy l'Etoile, France) was used at 2.5 μg/ml for cohort 2.

Techniques: Expressing