Human Plexin D1 Antibody

Detection of Human Plexin D1 by Western Blot. Western blot shows lysates of JAR human choriocarcinoma cell line, IMR-32 human neuroblastoma cell line, and Jurkat human acute T cell leukemia cell line. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human Plexin D1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4160) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Plexin D1 at approximately 250 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Plexin D1 in Human peripheral blood monocytes by Flow Cytometry. Human peripheral blood monocytes were stained with Goat Anti-Human Plexin D1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4160, filled histogram) or control antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107).

Plexin D1 in Human Melanoma. Plexin D1 was detected in immersion fixed paraffin-embedded sections of human melanoma using Goat Anti-Human Plexin D1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4160) at 3 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to plasma membranes. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Proliferation Inhibited by Semaphorin 3E and Neutralization by Human Plexin D1 Antibody. Recombinant Human Semaphorin 3E (Catalog # 3239-S3) inhibits proliferation in the HUVEC human umbilical vein endothelial cells in a dose-dependent manner (orange line). Plexin D1 mediated inhibition elicited by Recombinant Human Semaphorin 3E (5 ug/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Human Plexin D1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4160). The ND₅₀ is typically 0.5-2 ug/mL.

Detection of Plexin D1 by Western Blot The absence of Mtss1 does not affect medium spiny neuron (MSN) survival, dendritic arborization, and Plexin-D1 expression during striatonigral pathway development.(A) Immunohistochemistry staining for cleaved caspase 3 (CC3) in the striatum of wild-type (WT) or Mtss1 conditional knockout (cKO) mice. The white dotted boxes on the left images are shown in the inset images on the right at a better resolution. Scale bar, 25 μm. (B) Quantification of cell death by the number of CC3-positive cells in a 1 mm2 area covering the dorsal part of the striatum in WT or Mtss1 cKO mice. Error bars, mean ± SEM; ns p>0.05 by Mann‒Whitney test; WT, n = 20; Mtss1 cKO mice, n = 20 (five sections/mouse). (C) Representative images of Golgi staining at low (top panels) and high (bottom panels) magnification. Scale bars, 100 μm. Sholl analysis of dendritic morphology (D) and dendritic length (E) performed by using Neurolucida360 in 3D analysis. Error bars, mean ± SEM; ns p>0.05 by Student’s t-test; WT, n = 12, and Mtss1 cKO mice, n = 15, from three mice. (F, G) Western blot images and quantification of Plexin-D1 expression in the striatum of WT or Mtss1 cKO mice at P5. Error bars, mean ± SEM; *p<0.05 by Student’s t-test; WT mice, n = 3, and Mtss1 cKO mice, n = 4. (H) Plexin-D1 expression in MSNs obtained from the striatum of WT or Mtss1 cKO mice at P0 and measured at DIV6 in culture. (I) Quantification of the western blots shown in (H). Error bars, mean ± SEM; *p<0.05, by Student’s t-test; n = 3 for WT, n = 3 for KO in three independent experiments. Figure 7—figure supplement 3—source data 1.Raw uncropped western blot & gel images. Western blots shown in Figure 7—figure supplement 3F and H.Raw uncropped western blot & gel images. Western blots shown in Figure 7—figure supplement 3F and H. Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Plexin D1 by Western Blot The absence of Mtss1 does not affect medium spiny neuron (MSN) survival, dendritic arborization, and Plexin-D1 expression during striatonigral pathway development.(A) Immunohistochemistry staining for cleaved caspase 3 (CC3) in the striatum of wild-type (WT) or Mtss1 conditional knockout (cKO) mice. The white dotted boxes on the left images are shown in the inset images on the right at a better resolution. Scale bar, 25 μm. (B) Quantification of cell death by the number of CC3-positive cells in a 1 mm2 area covering the dorsal part of the striatum in WT or Mtss1 cKO mice. Error bars, mean ± SEM; ns p>0.05 by Mann‒Whitney test; WT, n = 20; Mtss1 cKO mice, n = 20 (five sections/mouse). (C) Representative images of Golgi staining at low (top panels) and high (bottom panels) magnification. Scale bars, 100 μm. Sholl analysis of dendritic morphology (D) and dendritic length (E) performed by using Neurolucida360 in 3D analysis. Error bars, mean ± SEM; ns p>0.05 by Student’s t-test; WT, n = 12, and Mtss1 cKO mice, n = 15, from three mice. (F, G) Western blot images and quantification of Plexin-D1 expression in the striatum of WT or Mtss1 cKO mice at P5. Error bars, mean ± SEM; *p<0.05 by Student’s t-test; WT mice, n = 3, and Mtss1 cKO mice, n = 4. (H) Plexin-D1 expression in MSNs obtained from the striatum of WT or Mtss1 cKO mice at P0 and measured at DIV6 in culture. (I) Quantification of the western blots shown in (H). Error bars, mean ± SEM; *p<0.05, by Student’s t-test; n = 3 for WT, n = 3 for KO in three independent experiments. Figure 7—figure supplement 3—source data 1.Raw uncropped western blot & gel images. Western blots shown in Figure 7—figure supplement 3F and H.Raw uncropped western blot & gel images. Western blots shown in Figure 7—figure supplement 3F and H. Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Plexin D1 by Western Blot In cultured medium spiny neurons (MSNs), Mtss1 expression is directly regulated by Sema3E-Plexin-D1 signaling through the AKT pathway.(A) WB images showing Mtss1 expression in MSNs derived from the striatum of wild-type (WT) or Plxnd1 conditional knockout (cKO) mice at P0 & measured at DIV3 & DIV6 in culture. (B) Quantification of band intensity in (A). Two-way ANOVA with Tukey’s post hoc correction for multiple comparisons; n = 3. (C) Mtss1 expression in MSNs obtained from the striatum of WT or Sema3e KO mice at P0 & measured at DIV6 in culture. (D) Quantification of the blots shown in (C). Student’s t-test; n = 5 for WT, n = 5 for KO in five independent experiments. (E) Schematic illustration of the experimental strategy for Sema3E-ligand or MK2206, an AKT inhibitor treatment in MSN culture. (F, G) WB images showing Mtss1 expression after AP-Sema3E (2 nM) treatment in cultured MSNs derived from Sema3e KO mice or Plxnd1 cKO mice. (H, I) Quantification of (F, G). Student’s t-test; AP, n = 4, AP-sema3E, n = 4 for sema3e KO mice, AP n = 4, AP-sema3E n = 4 for Plxnd1 cKO mice in three independent experiments. (J, K) WB to analyze the expression of Mtss1 & Plexin-D1 after MK2206 (100 nM) treatment in cultured MSNs & subsequent quantification for band intensity (L, M). Student’s t-test; n = 6 for sham, n = 6 for MK2206 in six independent experiments. (N O) WB image & analysis showing Mtss1 expression in Sema3e knockout MSNs treated with MK2206 after incubation with AP-Sema3E. Two-way ANOVA with Tukey’s post hoc correction for multiple comparisons; n = 5 in five independent experiments. Error bars, mean ± SEM; *p<0.05, **p<0.01, ***p<0.001 by indicated statistical tests. Figure 2—source data 1.WBs shown in Figure 2A, C, F, G, J, K, & N.WBs shown in Figure 2A, C, F, G, J, K, & N. Image collected & cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Plexin D1 by Immunohistochemistry No Mtss1 was found in endothelial cells at E14.5, and no vascular defects were observed in Mtss1-conditional knockout (KO) mice.(A) Fluorescence in situ hybridization for Plxnd1 mRNA (green) and Mtss1 mRNA (red) in the brain and dorsal trunk at E14.5. White dotted boxes are shown in the inset image on the bottom. Scale bars, 100 μm for brain, 50 μm for dorsal trunk. (B) 3D vascular reconstruction analysis images after CD31 immunostaining and tissue clearing obtained using multifunctional fast confocal microscopy Dragonfly 502w. (C) Western blotting to analyze Mtss1 expression after AP-Sema3E (2 nM) treatment in human umbilical vein endothelial cells (HUVECs) or human cortical microvessel endothelial cells (HCMEC/D3). Error bars, mean ± SEM; ns p>0.05 by Mann‒Whitney test; AP, n = 4, AP-Sema3E, n = 4 for HUVECs, ns p>0.05 by Student’s t-test; AP, n = 4, AP-Sema3E, n = 4 for HCMEC/D3s in four dependent experiments. Figure 8—figure supplement 1—source data 1.Western blots shown in Figure 8—figure supplement 1C.Western blots shown in Figure 8—figure supplement 1C. Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Plexin D1 by Western Blot In cultured medium spiny neurons (MSNs), Mtss1 expression is directly regulated by Sema3E-Plexin-D1 signaling through the AKT pathway.(A) WB images showing Mtss1 expression in MSNs derived from the striatum of wild-type (WT) or Plxnd1 conditional knockout (cKO) mice at P0 & measured at DIV3 & DIV6 in culture. (B) Quantification of band intensity in (A). Two-way ANOVA with Tukey’s post hoc correction for multiple comparisons; n = 3. (C) Mtss1 expression in MSNs obtained from the striatum of WT or Sema3e KO mice at P0 & measured at DIV6 in culture. (D) Quantification of the blots shown in (C). Student’s t-test; n = 5 for WT, n = 5 for KO in five independent experiments. (E) Schematic illustration of the experimental strategy for Sema3E-ligand or MK2206, an AKT inhibitor treatment in MSN culture. (F, G) WB images showing Mtss1 expression after AP-Sema3E (2 nM) treatment in cultured MSNs derived from Sema3e KO mice or Plxnd1 cKO mice. (H, I) Quantification of (F, G). Student’s t-test; AP, n = 4, AP-sema3E, n = 4 for sema3e KO mice, AP n = 4, AP-sema3E n = 4 for Plxnd1 cKO mice in three independent experiments. (J, K) WB to analyze the expression of Mtss1 & Plexin-D1 after MK2206 (100 nM) treatment in cultured MSNs & subsequent quantification for band intensity (L, M). Student’s t-test; n = 6 for sham, n = 6 for MK2206 in six independent experiments. (N O) WB image & analysis showing Mtss1 expression in Sema3e knockout MSNs treated with MK2206 after incubation with AP-Sema3E. Two-way ANOVA with Tukey’s post hoc correction for multiple comparisons; n = 5 in five independent experiments. Error bars, mean ± SEM; *p<0.05, **p<0.01, ***p<0.001 by indicated statistical tests. Figure 2—source data 1.WBs shown in Figure 2A, C, F, G, J, K, & N.WBs shown in Figure 2A, C, F, G, J, K, & N. Image collected & cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Plexin D1 by Immunohistochemistry No significant alteration in the expression of vsv-Plexin-D1 or Mtss1-myc or Mtss1 delta I-BAR-myc in the medium spiny neuron (MSN) soma.(A) Representative image of Immunocytochemistry for vsv-Plexin-D1 (red), Mtss1-myc or Mtss1 delta I-BAR -myc (green) in the cell body of cultured MSNs transfected with vsv-Plexin-D1 and Mtss1-myc or Mtss1 delta I-BAR-myc, using Mtss1-null mice as a background. (B) Quantification of the fluorescence intensity in the cell body of (A). Error bars, mean ± SEM; ns p>0.05 by two-way ANOVA with Tukey’s post hoc correction for multiple comparisons; vsv-Plexin-D1+Mtss1 myc, n = 5, and vsv-Plexin-D1+Mtss1 delta I-BAR-myc, n = 5. Scale bar, 5 μm. Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Plexin D1 by Western Blot Expression of Mtss1 induces I-BAR domain-dependent morphological changes in COS7 cells, generating protrusions.(A) Western blot images showing that weakly expression of endogenous Mtss1 was not altered by overexpression of Plexin-D1 with or without Sema3E in COS7 cells. Asterisk indicates nonspecific band. (B) Quantification of the band intensity in (A). Error bars, mean ± SEM; ns p>0.05 by two-way ANOVA with Bonferroni’s post hoc correction for multiple comparisons; n = 3. (C) Schematics describing the full-length construct of Mtss1-myc and its deletion mutant constructs (Mtss1 delta I-BAR-myc, Mtss1 delta WH2-myc, and I-BAR-myc). (D) Immunocytochemistry images taken after overexpression of each construct. Constructs show the I-BAR domain leading to diverse cell protrusion morphologies. Some of the protrusions were excessively spiked or thin and long (arrowheads). Overexpression of the I-BAR domain only (I-BAR-myc) can induce extreme protrusion structures. Scale bar, 20 μm.Figure 3—figure supplement 1—source data 1.Western blots shown in Figure 3—figure supplement 1A.Western blots shown in Figure 3—figure supplement 1A. Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Plexin D1 by Western Blot Mtss1 expression alters Plexin-D1 localization to the protrusion structure in COS7 cells without affecting its endocytosis or Sema3E binding.(A) Cell surface biotinylation and subsequent Western blot analysis to analyze the surface localization of Plexin-D1 in COS7 cells. Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Plexin D1 by Immunohistochemistry Mtss1 facilitates Plexin-D1 transport to the growth cone in cultured Drd1a-positive medium spiny neurons (MSNs).(A) Immunocytochemistry for Mtss1-myc or Mtss1 delta I-BAR -myc (green), vsv-Plexin-D1 (red) in the axons of MSNs transfected with vsv-Plexin-D1 and Mtss1-myc or Mtss1 delta I-BAR-myc, using Mtss1-null mice as a background. The images were acquired using structured illumination microscopy (N-SIM). Scale bar, 5 μm. Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Plexin D1 by Western Blot Mtss1 facilitates Plexin-D1 transport to the growth cone in cultured Drd1a-positive medium spiny neurons (MSNs). Quantification of colocalization by Pearson’s correlation coefficient calculated using Costes’ randomized pixel scrambled image method. Student’s t-test; vsv-Plexin-D1+Mtss1-myc, n = 21, and vsv-Plexin-D1+Mtss1 delta IBAR-myc, n = 14. Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Plexin D1 by Immunohistochemistry Mtss1 expression alters Plexin-D1 localization to the protrusion structure in COS7 cells without affecting its endocytosis or Sema3E binding. (F) Immunocytochemistry for vsv-Plexin-D1 (red), Mtss1-myc (green), and F-actin (gray) in COS7 cells. Images were obtained by structured illumination microscopy (N-SIM). White arrows (top) indicate colocalized Plexin-D1 and Mtss1 in the protrusion structure. White arrowheads (middle) indicate high Mtss1 levels localized in cell protrusions without Plexin-D1. Yellow arrows (bottom) indicate the normal cell surface with Mtss1 delta I-BAR but no Plexin-D1 colocalization. Scale bar, 5 μm. Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Plexin D1 by Western Blot Sema3E-Plexin-D1 signaling induces Mtss1 expression selectively in developing striatonigral projecting neurons. (K) Western blot images showing the expression of Mtss1 and Plexin-D1 in the striatum of WT or Plxnd1 cKO mice at different developmental stages ranging from embryonic day 16.5 (E16.5) to postnatal day 0 (P0). Image collected and cropped by CiteAb from the following open publication (https://elifesciences.org/articles/96891), licensed under a CC-BY license. Not internally tested by R&D Systems.