Mouse Netrin-1 Antibody

Detection of Human Netrin-1 by Western Blot

UNC5A and UNC5C induce caspase-3 activation through DAPK1/PR65b during UPR. (A) Quantification of netrin-1–receptor expression by qRT-PCR (mean + SEM, n = 3). (B–F) Identification of the implicated netrin-1 receptors. HepaRG cells were transfected with siRNAs and treated with DTT or not for 4 hours (mock). (B) Assessment of XBP1 mRNA splicing by RT-PCR. Representative result, n = 3. (C) Assessment of netrin-1 protein knockdown by immunoblotting. Representative result, n = 3. (D and E) Assessment of transcript knockdown efficiencies. Graphs indicate (D) UNC5A and (E) UNC5C mRNA levels in siRNA-treated cells in comparison with control siRNA-treated cells (mean + SEM; n = 3; Mann–Whitney test; P < .05). (F) Caspase-3 activation is reversed by UNC5A or UNC5C knockdown after UPR induction. Graph indicates caspase-3 activity ratio of DTT vs untreated cells for each condition (mean + SEM; n = 3; Mann–Whitney test; P < .05). (G–J) Identification of the downstream signaling pathway. HepaRG cells were transfected with siRNAs and treated with DTT or not for 4 hours (mock). (G) Assessment of XBP1 mRNA splicing by RT-PCR. Representative result, n = 3. (H) Evaluation of netrin-1, DAPK1, and PR65 beta depletion by immunoblotting. Representative result, n = 3. (I) Caspase-3 activation is reversed by DAPK1 or PR65 beta knockdown. Graph indicates the caspase-3 activity ratio for each condition (mean + SEM; n = 3; Mann–Whitney test; P < .05). (J) PP2A activity is increased by netrin-1 depletion and reversed by reduced expression of PR65 beta. Graph indicates PP2A activity ratio for each condition (mean + SEM; n = 3; Mann–Whitney test; P < .05). *,**, or *** refer to statistical analyses. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28174720), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Netrin-1 by Western Blot

Netrin-1 protects against cell death during UPR. (A–H) Netrin-1 depletion. (A–E) HepaRG cells were transfected with netrin-1 siRNA, treated with DTT, and harvested in a time course assay. (A) Netrin-1 protein knockdown by siRNA was validated by immunoblot (representative result, n = 3). (B) XBP1 splicing was confirmed by RT-PCR (representative result, n = 3). (C) UPR increases the dependence of cells toward netrin-1 for survival. Graphs indicate the difference in cell death (in percentages) (mean + SEM; n = 3; Mann–Whitney test; P < .05). (D) UPR increases the sensitivity of cells toward netrin-1 for caspase-3 activation. Graphs indicate caspase-3 activity ratios (mean + SEM; n = 3; Mann–Whitney test; P < .05). (E) Netrin-1 depletion increases apoptosis. Graph indicates apoptotic cell death ratio by propidium iodide staining and flow cytometry (mean + SEM; n = 3; Mann–Whitney test; P < .05). (F–H) Netrin-1 inhibition using a neutralizing antibody. HepaRG cells were seeded and treated with a control (H4) or anti–netrin-1 antibody (2F5) the same day. Cells were treated with DTT 3 days after addition and harvested after the indicated time points. (F) Assessment of XBP1 mRNA splicing (representative result, n = 3). (G) Netrin-1 neutralization enhances caspase-3 activity. Graph indicates the caspase-3 activity ratios between 2F5 and H4-treated cells (mean + SEM; n = 3; Mann–Whitney test; P < .05). (H) Netrin-1 depletion increases apoptosis. Graph indicates the apoptotic cell death ratio as assessed after propidium iodide staining and flow cytometry of netrin-1–depleted and control cells (mean + SEM; n = 3; Mann–Whitney test; P < .05). (I–M) Netrin-1 forced expression. HepaRG cells were transfected with control (VR1-HA) and netrin-1 (netrin-1–HA) vectors, treated with DTT, and harvested. (I) Netrin-1 expression was assessed by immunoblotting. Representative result, n = 3. (J) XBP1 mRNA splicing was quantified by RT-PCR at the indicated time points. Representative result, n = 3. (K) UPR increases the sensitivity of cells toward netrin-1 for protection against cell death in time course assays. Graph indicates the difference in cell death (in percentages) between netrin-1–overexpressing cells and control cells (mean + SEM; n = 3; Mann–Whitney test; P < .05). (L) UPR increases the sensitivity of cells toward netrin-1 for caspase-3 activation in time course assays. Graph indicates the ratios of caspase-3 activation levels among netrin-1–overexpressing and control cells (mean + SEM; n = 3; Mann–Whitney test; P < .05). (M) Netrin-1 overexpression decreases apoptosis in a time course assay. Graph indicates the apoptotic cell death ratio as assessed after propidium iodide staining and flow cytometry of netrin-1–overexpressing and control cells (mean + SEM; n = 3; Mann–Whitney test; P < .05). *,**, or *** refer to statistical analyses. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28174720), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Netrin-1 by Western Blot

Netrin-1 reverts UPR-induced caspase-3 activation in netrin-1 transgenic mice. Netrin-1 (FLAG-tagged) transgenic mice or control littermates were treated with Tamoxifen (Tamox), injected with PBS, or 1 mg/kg Tu and killed 24 hours after treatment. (A) Evaluation of netrin-1 protein overexpression by anti-FLAG immunoblotting. (B) Evaluation of netrin-1 protein overexpression by immunohistochemistry. Representative result, n = 5 (wt/tamox/PBS), n = 6 (wt/tamox/Tu), n = 3 (netrin-1/tamox/PBS), n = 5 (netrin-1/tamox/Tu for the whole figure). Background level was assessed using an isotype control antibody. Magnification: ×20. (C) Liver pictures 24 hours after treatment. (D) Evaluation of eIF2 alpha phosphorylation by immunoblotting. (E) Caspase-3 activation after Tu treatment is reversed in netrin-1–expressing transgenic mice. Graph indicates fold changes in caspase-3 activity compared with control mice (mean + SEM; Mann–Whitney test; P < .05). (F) The number of apoptotic cells is decreased in netrin-1 transgenic mice after Tu treatment. Graph indicates TUNEL positive cells/mm2 for each group (mean + SEM; Mann–Whitney test; P < .05). See also Supplementary Figure 6. *,**, or *** refer to statistical analyses. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28174720), licensed under a CC-BY license. Not internally tested by R&D Systems.