unc5b antibody  (R&D Systems)

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: Unc5B expression in endothelial cells and mural cells of the developing retina. (a) UMAP plot of retinal cells . (b) Unc5B dot plot expression levels and frequency among retina cell clusters. Color scale: red: high expression; green: low expression. (c) UMAP plot of EC sub-clusters. (d) Unc5B dot plot expression levels and frequency among ECs subclusters. Color scale: red: high expression; green: low expression. (e-h) Immunofluorescence staining and confocal imaging on whole-mount P5 or P12 retinas with the indicated antibodies.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Expressing, Immunofluorescence, Staining, Imaging

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: (a,b) Violin plots of the expression of Unc5B in retina cells (a) or in endothelial cell subclusters (b) . (c) Dot plot expression levels and frequency of Unc5B ligands among retina cell clusters . Color scale: yellow: high expression; dark blue: low expression.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Expressing

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: (a) Unc5B gene deletion strategy using tamoxifen injection in P5 neonates. (b-e) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (f) Quantification of GFAP+ astrocytes and PDGFRβ+ pericyte vascular coverage in P5 retinas. All data are shown as mean+/-SEM. Mann-Whitney U test was performed for statistical analysis between two groups.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Immunofluorescence, Staining, Imaging, MANN-WHITNEY

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: Endothelial Unc5B regulates retinal angiogenesis and BRB formation. (a) Unc5B gene deletion strategy using tamoxifen injection in P5 neonates. (b) IB4 immunofluorescence staining and confocal imaging on whole-mount P5 retinas. (c) Quantification of IB4+ vascular outgrowth and density in P5 retinas. (d) Unc5B gene deletion strategy using tamoxifen injection in P12 neonates. (e) Immunofluorescence staining of IB4+ vessel and confocal imaging on whole-mount P12 retinas. Superficial, intermediate and deep plexus are color-coded. (f) Quantification of superficial, intermediate and deep plexus vascular density in P12 retinas. (g) Unc5B gene deletion strategy using tamoxifen injection in P5 neonates. (h) Confocal imaging on whole-mount P5 retinas, 2h after i.p. cadaverine injection. (i) quantification of cadaverine leakage. (j) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies, 2h after i.p. cadaverine injection. (k) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (l,m) Western blot (l) and quantification (m) of P5 retina protein extracts. (n) Unc5B gene deletion strategy using tamoxifen injection in P12 neonates. (o) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies, 10min after i.v. sulfo-NHS-biotin injection. All data are shown as mean+/-SEM. Two-sided Mann-Whitney U test was performed for statistical analysis.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Immunofluorescence, Staining, Imaging, Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: Endothelial Unc5B regulates BRB permeability via Norrin/β-catenin signaling. (a) Unc5B gene deletion strategy using tamoxifen injection in P5 neonates. (b,c) Western blot (b) and quantification (c) of P5 retina protein extracts. (d) Unc5B and Ctnnb1 gene deletion strategy using tamoxifen injection in P5 retinas. (e) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (f) Unc5B and Ctnnb1 gene deletion strategy using tamoxifen injection in P12 retinas. (g) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies. (h) Unc5B gene deletion and Ctnnb1 flex/3 gene overexpression strategy using tamoxifen injection in P5 retinas. (i) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (j) Unc5B gene deletion and Ctnnb1 flex/3 gene overexpression strategy using tamoxifen injection in P12 retinas. (k) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies, 10min after i.v. sulfo-NHS-biotin injection. All data are shown as mean+/-SEM. Two-sided Mann-Whitney U test was performed for statistical analysis.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Permeability, Injection, Western Blot, Immunofluorescence, Staining, Imaging, Over Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: (a) Unc5B gene deletion strategy using tamoxifen injection in P12 neonates. (b,c) Western blot (b) and quantification (c) of P5 retina protein extracts. (d,e) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. All data are shown as mean+/-SEM. Mann-Whitney U test was performed for statistical analysis between two groups.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Western Blot, Immunofluorescence, Staining, Imaging, MANN-WHITNEY

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: Netrin-1 regulates BRB development and integrity. (a) Ntn1 gene deletion strategy using tamoxifen injection in P5 neonates. (b) IB4 immunofluorescence staining and confocal imaging on whole-mount P5 retinas. (c) Quantification of IB4+ vascular outgrowth and density in P5 retinas. (d) Ntn1 gene deletion strategy using tamoxifen injection in P12 neonates. (e) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies. (f) Netrin1 binding to Unc5B blockade strategy using anti-Unc5B-3 antibody. (g) Confocal imaging on P12 retinas i.p. injected with anti-Unc5B-3 (2h, 10mg/kg) followed by i.v. sulfo-NHS-biotin injection for 10min. (h) Ntn1 gene deletion strategy using tamoxifen injection in P5 neonates. (i,j) Western blot (i) and quantification (j) of P5 retina protein extracts. (k) Ntn1 gene overexpression strategy using tamoxifen injection in P5 neonates. (l) qPCR analysis of P5 retina mRNA extracts. (m) IB4 immunofluorescence staining and confocal imaging on whole-mount P5 retinas. (n) Quantification of IB4+ vascular outgrowth and density in P5 retinas. (o) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (p) Ntn1 gene overexpression strategy using tamoxifen injection in P5 neonates. (q) Confocal imaging on P5 retinas i.v. injected with sulfo-NHS-biotin for 10mim. All data are shown as mean+/-SEM. Two-sided Mann-Whitney U test was performed for statistical analysis.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Immunofluorescence, Staining, Imaging, Binding Assay, Western Blot, Over Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: Mural cell Unc5B contributes to retinal angiogenesis. (a) Global or Pericyte Unc5B gene deletion strategy using tamoxifen injection. (b,c) Survival curve after neonatal global (b) or pericyte (c) Unc5B gene deletion. (d) Immunofluorescence staining and confocal imaging on whole-mount P5 retinas with the indicated antibodies. (e,f) Quantification on IB4+ vascular outgrowth and density in P5 retinas. (g) Immunofluorescence staining of IB4+ vessel and confocal imaging on whole-mount P12 retinas. Superficial, intermediate and deep plexus were color-coded. (h,i) Quantification of superficial, intermediate and deep plexus vascular density in P12 retinas. (j,k) Immunofluorescence staining and confocal imaging on the vascular front of whole-mount P5 retinas with the indicated antibodies. All data are shown as mean+/- SEM. Mantel-cox test was performed for survival curve statistical analysis. Mann-Whitney U test was performed for statistical analysis between two groups. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Immunofluorescence, Staining, Imaging, MANN-WHITNEY

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: Mural cell Unc5B contributes to BRB regulation. (a) Unc5B gene deletion strategy using tamoxifen injection in P12 neonates. (b) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies, 10min after i.v. sulfo-NHS-biotin injection. (c) Unc5B gene deletion strategy using tamoxifen injection in P12 neonates. (d) Immunofluorescence staining and confocal imaging on whole-mount P12 retinas with the indicated antibodies. (e) Unc5B gene deletion strategy using tamoxifen injection in P5 neonates. (f,g) Western blot (f) and quantification (g) of P5 retina protein extracts. All data are shown as mean+/-SEM. Mann-Whitney U test was performed for statistical analysis between two groups.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Injection, Immunofluorescence, Staining, Imaging, Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

doi: 10.1101/2023.01.21.525006

Figure Lengend Snippet: Netrin-1 and Norrin cooperate to induce β-catenin signaling (a-c) Western blot (a) of mouse brain EC protein extracts treated for the indicated times (min) with Netrin-1, Norrin, or a combination of both. (b,c) Quantification of LRP5 phosphorylation levels over time (d) Mouse brain ECs were transfected with the β-catenin transcriptional luciferase reporter TOPflash or FOP flash control and stimulated with Netrin-1, Norrin, or a combination. Cells were harvested in 1x cell culture lysis reagent and luciferase activity was quantified using a plate-based luminometer. (e) CTRL IgG and Unc5B immunoprecipitation from cultured mouse brain ECs. (f) CTRL IgG and Unc5B immunoprecipitation on CTRL or Dlg1 SiRNA-treated mouse brain ECs. (g,h) Western blot (g) and quantification (h) of CTRL, Unc5B or Dlg1 SiRNA-transfected mouse brain ECs treated with Netrin-1 (250ng/ml). All data are shown as mean+/-SEM. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups.

Article Snippet: Protein lysates were harvested using a magnetic separator (Invitrogen) and were incubated overnight at 4°C under gentle rotation with 10ug of Unc5B antibody (R&D, AF1006) or control IgG.

Techniques: Western Blot, Transfection, Luciferase, Cell Culture, Lysis, Activity Assay, Immunoprecipitation

Journal: Science Advances

Article Title: The FLRT3-UNC5B checkpoint pathway inhibits T cell–based cancer immunotherapies

doi: 10.1126/sciadv.adj4698

Figure Lengend Snippet: ( A and B ) Human PBMCs from three healthy donors were activated with CD3 + CD28 Abs, and FLRT3 binding partner expression was analyzed on CD8 + T cells on days 0 (naïve), 3, and 4 (activated). (A) Representative histograms for UNC5B and LPHN1 staining on day 3, and (B) quantification of % CD8 + T cell expression. Each dot represents one donor, and error bars denote SD. ( C ) Representative dot plots of UNC5B and checkpoint receptor expression in CD8 + T cells from PBMCs activated with CD3 Abs for 10 days. ( D ) Illustration showing Jurkat-NG reporter system. ( E and F ) Jurkat-NG cells were transduced with LPHN3 or UNC5B and cocultured with EV- or FLRT3-expressing 293T-OKT3 cells for 16 hours, followed by GFP quantification by flow cytometry. Quantification of GFP MFI in Jurkat-NG cells with or without (E) UNC5B or (F) LPHN3 overexpression. ( G ) UNC5B–Jurkat-NG cells were cocultured with 293T-OKT3 cells expressing FLRT3, PD-L1, or CD80 for 16 hours, and NF-κB activity (GFP) was measured by flow cytometry. Quantification of GFP is shown. ( H and I ) UNC5B–Jurkat-NG cells were activated with (H) CD3 + CD28 Abs or (I) soluble CD3 Abs on control Fc of FLRT3 Fc–coated plates for 16 hours, and NF-κB activity (GFP) was measured by flow cytometry. Quantification of GFP levels is shown. For (E) to (I), each data point represents one technical replicate and error bars denote +SD. Data representative of at least two independent experiments. Statistical significance was determined for (E), (F), (H), and (I) by unpaired t test and for (G) by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. For all the data, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human UNC5B antibody , Abcam , ab104871.

Techniques: Binding Assay, Expressing, Staining, Transduction, Flow Cytometry, Over Expression, Activity Assay

Journal: Science Advances

Article Title: The FLRT3-UNC5B checkpoint pathway inhibits T cell–based cancer immunotherapies

doi: 10.1126/sciadv.adj4698

Figure Lengend Snippet: ( A ) Quantification of GFP–NF-κB in UNC5B-Jurkat-NG cells following 16 hours of coculture with EV or FLRT3-293T-OKT3 cells, and NP591 or isotype. Data representative of two experiments. ( B ) Four-day activated human PBMCs from three donors were cocultured with SKOV3 cells with CD3 + CD28 Abs in the presence of NP591 or isotype Ab. Three days later, IFN-γ was measured by ELISA. ( C and D ) PBMCs from three healthy donors (responders) were cocultured with donor PBMCs (stimulators) at a 1:1 ratio for 7 days in the presence of irradiated SKOV3 cells. Isotype control or NP591 was added in the cultures on days 0 and 3. On day 7, supernatants were harvested for IFN-γ (C) and TNF-α (D) analysis by ELISA. Bar graph data points in (A) to (D) represent one replicate, and error bars denote SD. ( E ) FLRT3-293T cells were admixed with human PBMCs and injected in mice intradermally. Mice were treated with NP591 anti–PD-1, NP591 and anti–PD-1 combo, or isotype control intraperitoneally starting on day 7 every 2 to 3 days × 3 weeks, then once per week. n = 12 per group. ( F ) Human PBMCs were injected intravenously followed by intradermal injection of SKOV3 cells 1 day later. Mice were treated with NP591 or isotype control intraperitoneally starting on day 7 every 2 to 3 days × 2 weeks, then once per week. N = 9 for isotype, n = 10 for NP591. Error bars denote SEM in (E) and (F). Data representative of two independent experiments. Statistical significance was determined for (A) by unpaired t test, for (B) by one-way ANOVA with Tukey’s post hoc test for multiple comparisons, for (C) and (D) by two-way ANOVA with Tukey’s post hoc test for multiple comparisons, and for (E) and (F) by paired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human UNC5B antibody , Abcam , ab104871.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Injection

Journal: Science Advances

Article Title: The FLRT3-UNC5B checkpoint pathway inhibits T cell–based cancer immunotherapies

doi: 10.1126/sciadv.adj4698

Figure Lengend Snippet: List of commercial FACS antibodies/reagents used for cell staining.

Article Snippet: Human UNC5B antibody , Abcam , ab104871.

Techniques: Staining, Purification

Journal: Science Advances

Article Title: The FLRT3-UNC5B checkpoint pathway inhibits T cell–based cancer immunotherapies

doi: 10.1126/sciadv.adj4698

Figure Lengend Snippet: List of binding ELISA-related proteins/antibodies.

Article Snippet: Human UNC5B antibody , Abcam , ab104871.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Cell reports

Article Title: Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling

doi: 10.1016/j.celrep.2023.113369

Figure Lengend Snippet: (A) RNA-seq analysis of primary and metastatic pancreatic cancer cell-line expression of Netrin-1 receptors. (B) Annexin V staining of primary Ink4a and metastatic Met38 cell lines transiently transfected with a non-targeting control siRNA or Ntn1 siRNA. n = 3 in each group. (C) (Left) Graphs of the migration and invasion analysis of serum starved Ink4a cells treated with recombinant Netrin-1. (Right) Representative images of fixed and stained cells are on the right. n = 10. Scale bars, 50 μm. (D) (Left) Graphs of the migration and invasion analysis of Met38 cells transiently transfected with a control or Ntn1 siRNA. (Right) Representative images of fixed and stained cells are on the right. n = 10. Scale bars, 50 μm. (E) 3D in vitro sprouting assay for Met38 control KD (a, b) or Ntn1 KD cells (c, d) and A1925 control (e, f) and Δ Unc5b (g, h) expressing cells. Netrin-1 knockdown was confirmed by Netrin-1 expression analysis (far right). Scale bars, 100 μm (a, c, e, g) and 20 μm (b, d, f, h). (F) (Top) Graph of the invasion analysis of Met38 cKO (control knockout) and Met38 Unc5b KO cells. Representative images of fixed and stained cells are below (scale bars, 50 μm), as well as the western blot showing Unc5b depletion in Unc5b KO cells. (G and H) H&E staining of livers after intrasplenic injection of Met38 control knockdown (KD) and Met38 Ntn1 KD cells (G) or Met38 cKO and Met38 Unc5b KO cells (H). Quantitation of tumor area was determined by VisioPharm software. n = 6/group. Scale bars, 50 μm. Statistical analysis was completed using two-tailed Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Other antibodies included CD9 (1:2000; clone EPR2949; cat #ab92726; Abcam); Unc5b (1:1000; clone D9M7Z; cat# 13851; Cell Signaling), mouse polyclonal flotillin-1 (1:500; cat #610820; BD Bioscience); CK17; cat# 606-540; Thermo Fisher), RARy1 (1:500; clone D3A4; cat# 8965, Cell Signaling), RXRβ (1:500; cat# ab221115; Abcam), RXRy (clone G-6, cat# sc-514134, Santa Cruz Biotechnology, actin (1:5000; clone H-6; cat# sc-376421; Santa Cruz), and Alix (1:1000; clone 3A9; cat # 2171; Cell Signaling).

Techniques: RNA Sequencing, Expressing, Staining, Transfection, Control, Migration, Recombinant, In Vitro, Knockdown, Knock-Out, Western Blot, Injection, Quantitation Assay, Software, Two Tailed Test

Journal: Cell reports

Article Title: Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling

doi: 10.1016/j.celrep.2023.113369

Figure Lengend Snippet: EVs containing Netrin-1 activate HSCs through both long-range (A) and possibly short-range communication upon dissemination in the liver (B). Upon activation by Netrin-1, HSCs dump their retinoic acid stores (C). The free retinoic acid is taken up by DTCs by their retinoic acid receptors (D), leading to upregulation of Netrin-1 directly and indirectly through Elf3 upregulation. Through autocrine signaling (E) by Unc5b receptors, newly arrived DTCs increase survival potential through Netrin-1 excretion. In addition, activated hepatic stellate cells deposit collagen into the microenvironment (F), which over time allows macrometastases to become established in the liver (G).

Article Snippet: Other antibodies included CD9 (1:2000; clone EPR2949; cat #ab92726; Abcam); Unc5b (1:1000; clone D9M7Z; cat# 13851; Cell Signaling), mouse polyclonal flotillin-1 (1:500; cat #610820; BD Bioscience); CK17; cat# 606-540; Thermo Fisher), RARy1 (1:500; clone D3A4; cat# 8965, Cell Signaling), RXRβ (1:500; cat# ab221115; Abcam), RXRy (clone G-6, cat# sc-514134, Santa Cruz Biotechnology, actin (1:5000; clone H-6; cat# sc-376421; Santa Cruz), and Alix (1:1000; clone 3A9; cat # 2171; Cell Signaling).

Techniques: Activation Assay

Journal: Cell reports

Article Title: Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling

doi: 10.1016/j.celrep.2023.113369

Figure Lengend Snippet: (A) RNA-seq analysis of primary and metastatic pancreatic cancer cell-line expression of Netrin-1 receptors. (B) Annexin V staining of primary Ink4a and metastatic Met38 cell lines transiently transfected with a non-targeting control siRNA or Ntn1 siRNA. n = 3 in each group. (C) (Left) Graphs of the migration and invasion analysis of serum starved Ink4a cells treated with recombinant Netrin-1. (Right) Representative images of fixed and stained cells are on the right. n = 10. Scale bars, 50 μm. (D) (Left) Graphs of the migration and invasion analysis of Met38 cells transiently transfected with a control or Ntn1 siRNA. (Right) Representative images of fixed and stained cells are on the right. n = 10. Scale bars, 50 μm. (E) 3D in vitro sprouting assay for Met38 control KD (a, b) or Ntn1 KD cells (c, d) and A1925 control (e, f) and Δ Unc5b (g, h) expressing cells. Netrin-1 knockdown was confirmed by Netrin-1 expression analysis (far right). Scale bars, 100 μm (a, c, e, g) and 20 μm (b, d, f, h). (F) (Top) Graph of the invasion analysis of Met38 cKO (control knockout) and Met38 Unc5b KO cells. Representative images of fixed and stained cells are below (scale bars, 50 μm), as well as the western blot showing Unc5b depletion in Unc5b KO cells. (G and H) H&E staining of livers after intrasplenic injection of Met38 control knockdown (KD) and Met38 Ntn1 KD cells (G) or Met38 cKO and Met38 Unc5b KO cells (H). Quantitation of tumor area was determined by VisioPharm software. n = 6/group. Scale bars, 50 μm. Statistical analysis was completed using two-tailed Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: UNC5B (D9M7Z) Rabbit mAb , Cell Signaling , 13851.

Techniques: RNA Sequencing, Expressing, Staining, Transfection, Control, Migration, Recombinant, In Vitro, Knockdown, Knock-Out, Western Blot, Injection, Quantitation Assay, Software, Two Tailed Test

Journal: Cell reports

Article Title: Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling

doi: 10.1016/j.celrep.2023.113369

Figure Lengend Snippet: EVs containing Netrin-1 activate HSCs through both long-range (A) and possibly short-range communication upon dissemination in the liver (B). Upon activation by Netrin-1, HSCs dump their retinoic acid stores (C). The free retinoic acid is taken up by DTCs by their retinoic acid receptors (D), leading to upregulation of Netrin-1 directly and indirectly through Elf3 upregulation. Through autocrine signaling (E) by Unc5b receptors, newly arrived DTCs increase survival potential through Netrin-1 excretion. In addition, activated hepatic stellate cells deposit collagen into the microenvironment (F), which over time allows macrometastases to become established in the liver (G).

Article Snippet: UNC5B (D9M7Z) Rabbit mAb , Cell Signaling , 13851.

Techniques: Activation Assay

Journal: Cell & Bioscience

Article Title: Hypofucosylation of Unc5b regulated by Fut8 enhances macrophage emigration and prevents atherosclerosis

doi: 10.1186/s13578-023-00959-y

Figure Lengend Snippet: Unc5b contributes to the formation of atherosclerotic plaques. A Representative Oil Red O-stained images of aortic valve sections; The intimal thickness of the aortic valve is shown by H&E staining; The aortic arch was stained with H&E to reveal the intimal thickness (bar=500 μm). B Quantitative image analysis of Figure A. C Serum lipid levels such as TC, TG, LDL-C, HDL-C in different groups. D Livers from ApoE −/− mice in different group were stained with H&E, and the number of vacuolized structure was characterized as maker to present the level of liver lipid and necrosis. E Atherosclerotic aortic sinus were collected from ApoE −/− mice and immunostaining assay was performed on the aortic to detect the expression of Unc5b as well as Fut8 in plaque (bar=200 μm, n=4). Unc5b (red), Fut8 (green), DAPI (blue); The content of macrophage (CD68 + ) in aortic valve plaques was determined by immunohistochemistry (bar=500 μm, n=4). F Quantification of Unc5b and Fut8 expression from immunostaining assay. Data are reported as the mean ± S.D. G Atherosclerotic aortic sinus were collected from ApoE −/− mice in Development group, Ad-NC, Ad-sh and Ad-oe group. The aortic sinus was stained for Unc5b (red), Fut8 (green), CD68 (blue) and their co-localization (yellow merge). Data are reported as the mean ± S.D. (n＞3, ****, p< 0.0001vs vehicle group, ###, p< 0.001 vs Ad-NC group, t-test). The mice in the Ad-NC group, Ad-sh group and Ad-oe group were given NC or Unc5bshRNA or Unc5b over-expression adenovirus injection into the caudal vein for four weeks.

Article Snippet: The anti-Unc5b antibody was purchased from CST (Danvers, USA).

Techniques: Staining, Immunostaining, Expressing, Immunohistochemistry, Over Expression, Injection

Journal: Cell & Bioscience

Article Title: Hypofucosylation of Unc5b regulated by Fut8 enhances macrophage emigration and prevents atherosclerosis

doi: 10.1186/s13578-023-00959-y

Figure Lengend Snippet: Unc5b suppresses migration of macrophage foam cells Raw 264.7 cells were subjected to various concentrations of ox-LDL (0, 24, 50 and 75 μg/ml) for 24 h, F-actin was stained with phalloidin-iFluor 488 (green), and the nuclei were counterstained with DAPI (blue), pseudopodia of macrophages were marked in white arrows ( A ), the number of cells on the dark side was identified by Transwell assay ( B ). ( C ) Western blot analysis of Unc5b protein levels in Raw 264.7 cells with Unc5b oe or Unc5b siRNA . Quantification of Unc5b expression from Western blotting. Data are reported as the mean ± S.D. (n＞3, * p< 0.05vs vehicle group, ## p< 0.01 vs Unc5b oe group, t-test). ( D ) Raw 264.7 macrophages were pretreated with Unc5bsiRNA or pcDNA3.1-Unc5b plasmid combined with ox-LDL for another 24 h, cells were stained by F-actin (green), DAPI (blue), and pseudopodia (marked in white). (E, F) Raw 264.7 cells were pretreated with empty plasmid or Unc5b siRNA or Unc5b oe , the number of cells on the dark side was identified by Transwell assay upon ox-LDL (50 μg/ml) treatment. Data are reported as the mean ± S.D. (bar=100 μm, n=4, *** , p< 0.001 vs ox-LDL 0 μg/mL group, ### , p< 0.001 vs ox-LDL 50 μg/mL, t-test)

Article Snippet: The anti-Unc5b antibody was purchased from CST (Danvers, USA).

Techniques: Migration, Staining, Transwell Assay, Western Blot, Expressing, Plasmid Preparation

Journal: Cell & Bioscience

Article Title: Hypofucosylation of Unc5b regulated by Fut8 enhances macrophage emigration and prevents atherosclerosis

doi: 10.1186/s13578-023-00959-y

Figure Lengend Snippet: Hypofucosylation of Unc5b regulated by Fut8 facilitates macrophage migration. A , C The interaction between Unc5b and AAL, LCA, PHAL, VVL, MAL1, SNA detected by IP assays after ox-LDL treatment for 24 h. B , D Quantification of Unc5b expression from IP assay. Data are reported as the mean ± S.D. (n＞3, t-test). E HEK293T cells were pretreated with pCMV-Unc5b-GFP plasmid or pCMV-Unc5bko222,347-GFP plasmid and pCMV-Fut8-mCherry plasmid and cotransfected with pCMV-Fut8-mCherry plasmid, followed by immunostaining. ER (blue), Fut8 (red), Unc5b (green), merge (yellow)(bar=20 μm, n=4). F The α-1,6-fucosylated protein level of Unc5b was detected by LCA in cells with Fut8eo or Fut8siRNA plasmid treatment and the quantification of protein expression was shown below the blot. Data are reported as the mean ± S.D. (n＞3, *, p< 0.05vs NC group, t-test).

Article Snippet: The anti-Unc5b antibody was purchased from CST (Danvers, USA).

Techniques: Migration, Expressing, Plasmid Preparation, Immunostaining

Journal: Cell & Bioscience

Article Title: Hypofucosylation of Unc5b regulated by Fut8 enhances macrophage emigration and prevents atherosclerosis

doi: 10.1186/s13578-023-00959-y

Figure Lengend Snippet: Unc5b affected macrophage migration through p-CDC42/p-PAK pathway. The expression of CDC42, p-CDC42 A as well as PAK, p-PAK B in Raw 264.7 cells after ox-LDL stimulation for 0-120 min were analyzed by Western blotting; C Quantification of p-CDC42 and p-PAK expression from Western blotting. Data are reported as the mean ± S.D. (n＞3, **, p< 0.01vs vehicle group, *p< 0.05 vs vehicle group, t-test). D CDC42, p-CDC42 and Rac123 protein levels in Raw 264.7 cells were analyzed by Western blotting. E Western blot analysis of PAK and p-PAK protein levels in Raw 264.7 cells. F Raw 264.7 cells were pretreated with CDC42 inhibitor or PAK inhibitor, and then the number of cells on the dark side was identified by Transwell assay upon ox-LDL (50 μg/ml) treatment. Also the effects of CDC42 inhibitor or PAK inhibitor on the formation of macrophage pseudopodium was detected by F-actin staining (bar=20 μm, n=4). G Raw 264.7 cells were pretreated with Unc5b siRNA or Unc5b oe combined with ox-LDL (50 μg/ml) stimulation, the Unc5b, CDC42 and PAK protein levels were determined by Western blotting. H Atherosclerotic aortic sinus were collected from ApoE −/− mice that was fed a Western diet (Development group) for 16 weeks, and also injected with Ad-NC, Ad-sh or Ad-oe for another 4 weeks. The aortic arch was stained for p-CDC42 and p-PAK (green). Data are reported as the mean ± S.D. (n=4, *, p< 0.05 ; ***, p< 0.001; t-test.)

Article Snippet: The anti-Unc5b antibody was purchased from CST (Danvers, USA).

Techniques: Migration, Expressing, Western Blot, Transwell Assay, Staining, Injection