ngf  (Alomone Labs)

Journal: Biomedicines

Article Title: p75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome

doi: 10.3390/biomedicines12112624

Figure Lengend Snippet: Expression of NGF and p75NTR fibroblasts derived from control individuals and Rett patients. ( A ) Total RNA was extracted from healthy control (HC-1) and Rett syndrome (RTT-1) fibroblasts, and the expression levels of ngf were measured by qRT-PCR. n = 3 biological replicates. Data represent means ± SD. ( B ) Immunofluorescence and respective quantitative analysis of NGF in HC and RTT. Cells were fixed in 4% PFA and stained with antibodies against NGF (red). DAPI (blue) was employed for nuclear counterstaining. n = 6 biological replicates. ( C ) qRT-PCR analysis of ngfr ( p75NTR ) in HC-1 and RTT-1 fibroblasts. n = 3 biological replicates. ( D ) Immunofluorescence and respective quantitative analysis of p75NTR immunoreactivity in HC-1 and RTT-1 cells. Cells were fixed in 4% PFA and stained with anti-p75NTR (red). DAPI (blue) was used to counterstain nuclei. n = 5 biological replicates. Data are expressed as mean ± SD. Statistical analysis was performed by using the Student’s unpaired t -test. Statistical significance is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. DMSO. Images were acquired using the Leica TCS SP8 confocal microscope and Leica Application Suite X (LAS X) software at 40× magnification. Scale bar: 50 µm.

Article Snippet: The anti-NGF and anti-p75NTR antibodies were validated using NGF (BLP-N240, Alomone labs, Jerusalem, Israel) and p75NTR (BLP-NT011 Alomone labs, Jerusalem, Israel) blocking peptides.

Techniques: Expressing, Derivative Assay, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Microscopy, Software

Journal: Biomedicines

Article Title: p75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome

doi: 10.3390/biomedicines12112624

Figure Lengend Snippet: Expression of NGF and p75NTR fibroblasts derived from control individuals and Rett patients. ( A ) Total RNA was extracted from healthy control (HC-1) and Rett syndrome (RTT-1) fibroblasts, and the expression levels of ngf were measured by qRT-PCR. n = 3 biological replicates. Data represent means ± SD. ( B ) Immunofluorescence and respective quantitative analysis of NGF in HC and RTT. Cells were fixed in 4% PFA and stained with antibodies against NGF (red). DAPI (blue) was employed for nuclear counterstaining. n = 6 biological replicates. ( C ) qRT-PCR analysis of ngfr ( p75NTR ) in HC-1 and RTT-1 fibroblasts. n = 3 biological replicates. ( D ) Immunofluorescence and respective quantitative analysis of p75NTR immunoreactivity in HC-1 and RTT-1 cells. Cells were fixed in 4% PFA and stained with anti-p75NTR (red). DAPI (blue) was used to counterstain nuclei. n = 5 biological replicates. Data are expressed as mean ± SD. Statistical analysis was performed by using the Student’s unpaired t -test. Statistical significance is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. DMSO. Images were acquired using the Leica TCS SP8 confocal microscope and Leica Application Suite X (LAS X) software at 40× magnification. Scale bar: 50 µm.

Article Snippet: The anti-NGF and anti-p75NTR antibodies were validated using NGF (BLP-N240, Alomone labs, Jerusalem, Israel) and p75NTR (BLP-NT011 Alomone labs, Jerusalem, Israel) blocking peptides.

Techniques: Expressing, Derivative Assay, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Microscopy, Software

Journal: Biomedicines

Article Title: p75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome

doi: 10.3390/biomedicines12112624

Figure Lengend Snippet: Effects of p75NTR modulation on the antioxidant response in RTT fibroblasts. Representative Western blot and densitometric analysis of ( A ) SOD1, ( B ) SOD2, ( C ) Catalase, ( D ) TrxR1, and ( E ) Gpx1 in healthy control fibroblasts (HC-1), Rett syndrome fibroblasts (RTT-1) and RTT fibroblasts treated with 0.1 μM of LM11A-31(RTT-1+LM) for 24 h. n = 3–5 biological replicates. GAPDH was used as a loading control. ( F ) Immunofluorescence and quantification of GSH immunoreactivity in HC-1, RTT-1, and RTT-1+LM experimental groups. Cells were fixed in 4% PFA and stained with antibodies against GSH (red). Nuclear staining was performed with DAPI (blue). n = 12 biological replicates. Data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test. Statistical significance is indicated as follows: ** p < 0.01; *** p < 0.001. “a” indicates statistical significance vs. HC-1; “b” indicates statistical significance vs. RTT-1. Images were acquired using the Leica TCS SP8 confocal microscope and Leica Application Suite X (LAS X) software at 40× magnification. Scale bar: 50 µm.

Article Snippet: The anti-NGF and anti-p75NTR antibodies were validated using NGF (BLP-N240, Alomone labs, Jerusalem, Israel) and p75NTR (BLP-NT011 Alomone labs, Jerusalem, Israel) blocking peptides.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Microscopy, Software

Journal: Biomedicines

Article Title: p75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome

doi: 10.3390/biomedicines12112624

Figure Lengend Snippet: p75NTR modulation reduces IL-6 and IL-8 expression in RTT fibroblasts. Representative immunofluorescence and respective quantitative analysis of ( A ) IL-6 immunoreactivity in control fibroblasts (HC-1), Rett syndrome fibroblasts (RTT-1), and RTT fibroblasts treated with LM11A-31(RTT-1+LM) at the dose of 0.1 μM for 24 h. Cells were fixed in 4% PFA and stained with antibodies against IL-6 (red). DAPI was used to counterstain nuclei. n = 6 biological replicates. Images were acquired using the Leica TCS SP8 confocal microscope and Leica Application Suite X (LAS X) software at 40× magnification. Scale bar: 50 µm. ( B ) ELISA on IL-6 in culture medium from HC-1, RTT-1, and RTT-1+LM groups treated as abovementioned. n = 4 biological replicates. ( C ) IL-8 immunoreactivity (red) and respective quantitative analysis performed on HC-1, RTT-1, and RTT-1+LM fibroblasts treated as in ( A ). DAPI was used for nuclear staining. n = 6 biological replicates. Scale bar: 50 µm. ( D ) ELISA on secreted IL-8 in conditioned medium from HC-1, RTT-1, and RTT-1+LM cells treated as abovementioned. n = 4 biological replicates. Data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test. Statistical significance is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001. “a” indicates statistical significance vs. HC-1; “b” indicates statistical significance vs. RTT-1.

Article Snippet: The anti-NGF and anti-p75NTR antibodies were validated using NGF (BLP-N240, Alomone labs, Jerusalem, Israel) and p75NTR (BLP-NT011 Alomone labs, Jerusalem, Israel) blocking peptides.

Techniques: Expressing, Immunofluorescence, Control, Staining, Microscopy, Software, Enzyme-linked Immunosorbent Assay