Human/Mouse/Rat Netrin-1 Antibody

Detection of Human, Mouse, and Rat Netrin‑1 by Western Blot.

Western blot shows lysates of IMR-32 human neuroblastoma cell line, bEnd.3 mouse endothelioma cell line, and C6 rat glioma cell line. PVDF membrane was probed with 0.5 µg/mL of Sheep Anti-Human/Mouse/Rat Netrin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6419) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for Netrin-1 at approximately 66 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.

Detection of Mouse Netrin-1 by Western Blot

MiDAC regulates neurite outgrowth via the SLIT3/ROBO3 and NTN1/UNC5B signaling pathways.(A) WB for signaling components of the SLIT3/ROBO3 and NTN1/DCC/UNC5B signaling axes from CM and total cell lysates of WT and Dnttip1 KO1 NE after 12 days of differentiation. To enrich SLIT3 and NTN1 from CM, IPs were performed with SLIT3 and NTN1 antibodies from CM of WT and Dnttip1 KO1 NE. Actin is the loading control for the total cell lysates. (B) Assay to rescue the neurite outgrowth defects in Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7–12 without or with preblocking of Dnttip1 KO1 NE with IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. MAP2 IF staining was performed after 12 days of differentiation. Nuclei were stained with DAPI. To facilitate analysis the neuronal cell body (blue) and its neurites were manually traced with ImageJ software and for each sample one traced neuron is displayed in the inlet. The white scale bar represents 50 µm. (C, D) Quantification of (C) neurite length and (D) the total number of neurites per neuron from the MAP2 IF staining in (B) using ImageJ. (C) Neurite length was divided into two categories of short neurites <50 µm (green box plots) and longer neurites ≥50 µm (white box plots). (C, D) The neurites of 200 neurons were assessed per sample. One-way ANOVA was performed throughout where ***, p≤0.001; and ns, p>0.05 is not significant.MiDAC regulates neurite outgrowth via the SLIT3/ROBO3 and NTN1/UNC5B signaling pathways.(A) Chamber assay to rescue the network formation defects of GNP-derived neurons that were supplemented with CM of Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7–12 without or with pre-blocking of GNP-derived neurons with control IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. TUBB3 IF staining was performed after 12 days of differentiation.

Detection of Netrin-1 by Western Blot

Expression of Netrin-1 and Neogenin in human melanoma cells. (A) Western blot results show different levels of Netrin-1 and Neogenin expression in human melanoma cell lines. The human neuroblastoma cell line SH-SY5Y and the normal human epidermal keratinocyte cell line HEKn were used as positive-expressing and poorly expressing controls for cellular expression of Netrin-1, respectively. (B) Results from immunofluorescence (IF) staining shows more intense expression of Neogenin in the aggressive melanoma cell lines C8161 and Sk-Mel28 compared to the poorly aggressive melanoma cell line UACC1273. SH-SY5Y was used as positive control for the IF staining for Neogenin. (Original magnification 20X). (C) The histogram shows the ratio of Neogenin to Netrin-1 expression determined from densitometric analysis of the respective WB bands and represented as a fold difference relative to the Neogenin-to-Netrin-1 ratio calculated for SH-SY5Y, which was used as control. These results clearly show that Neogenin expression relative to Netrin-1 is significantly greater in aggressive compared to poorly aggressive melanoma cells (* p < 0.05, n = 3). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36361539), licensed under a CC-BY license. Not internally tested by R&D Systems.