Mouse/Rat Neuropilin-1 Antibody

Detection of Recombinant Human, Mouse, and Rat Neuropilin‑1 by Western Blot. Western blot shows 25 ng of Recombinant Mouse Neuropilin-1 (Catalog # 5994-N1), Recombinant Rat Neuropilin-1 Fc Chimera (Catalog # 566-N1) and Recombinant Human Neuropilin-1 (Catalog # 3870-N1). PVDF Membrane was probed with 0.1 µg/mL of Goat Anti-Mouse/Rat Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF566) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). A specific band was detected for Neuropilin-1 at approximately 150 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Neuropilin-1 in bEnd.3 Mouse Cell Line by Flow Cytometry. bEnd.3 mouse endothelioma cell line was stained with Goat Anti-Rat Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF566, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram), followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0108).

Neuropilin‑1 in Embryonic Rat Spinal Cord. Neuropilin-1 was detected in immersion fixed frozen sections of embryonic rat spinal cord (15 d.p.c.) using 5 µg/mL Goat Anti-Rat Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF566) overnight at 4 °C. Tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counter-stained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Neuropilin‑1 in Rat Spinal Cord. Neuropilin-1 was detected in perfusion fixed frozen sections of rat spinal cord using Goat Anti-Rat Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF566) at 15 µg/mL overnight at 4 °C. Tissue was stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counter-stained with DAPI (blue). Specific staining was localized to the dorsal horn. View our protocol for Fluorescent IHC Staining of Frozen Tissue Sections.

Neuropilin‑1 in Rat Brain. Neuropilin-1 was detected in immersion fixed paraffin-embedded sections of rat brain (hippocampus) using Goat Anti-Mouse/Rat Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF566) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in neuronal cell bodies and projections. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Neuropilin‑1 in Rat Brain. Neuropilin-1 was detected in immersion fixed paraffin-embedded sections of rat brain (thalamus) using Goat Anti-Mouse/Rat Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF566) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in neuronal cell bodies and projections. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Mouse Neuropilin-1 by Western Blot Nrp1 influences Smad2/3 activation.(a–c) Representative confocal images of wt sprouts from EBs immunolabelled for pSmad2. The outline of the wt sprouts is indicated with a dashed line using the endogenous DsRED marker. DAPI staining (not shown) was used to mark nuclei (blue line). Sprouts from (a) untreated EBs, (b) treated with 2 ng ml−1 Tgf-beta for 1 h and(c) treated with 10 μM SB-421543 for 4 h. Scale bar, 13 μm. (d,e) Western blot analysis of proteins from P4 HUVEC transfected with control siRNA and NRP1 siRNA, with or without stimulation with 2 ng ml−1 TGF-beta for 1 h. Full western blots are shown in Supplementary Fig. 10. A representative blot of six is shown; P=0.0012 NRP1 siRNA compared with control. (f,g) Proteins from P4 HUVEC transfected with control-GFP and NRP1–GFP–His construct for 24 h, with or without stimulation with 2 ng ml−1 TGF-beta for 1 h were assessed for SMAD2 phosphorylation. Full western blots are shown in Supplementary Fig. 11. A representative blot of four is shown; P=0.0017 NRP1 overexpression compared with control. (e,g) Quantification of pSMAD2 protein normalized to SMAD2/3. (h–k) Western blot analysis of proteins from P4 HUVEC transfected with control siRNA and NRP1 siRNA, with or without stimulation with 2 ng ml−1 TGF-beta for 1 h. Full western blots are shown in Supplementary Figs 12 and 13. (i) Quantification of pSMAD2 protein normalized to SMAD2/3; P=0.0848 Nrp1 siRNA compared with control. (k) Quantification of pSMAD3 protein normalized to SMAD3; P=0.0181 NRP1 siRNA compared with control. (l,m) Western blot analysis of P4 HUVEC transfected with control siRNA and NRP1 siRNA, with or without stimulation with 10 ng ml−1 BMP9 for 15 and 30 min. Full western blots are shown in Supplementary Fig. 14. A representative blot of four is shown. (m) Quantification of pSMAD2/3 protein normalized to SMAD2; P<0.0011 NRP1 siRNA compared with control. All values represent mean±s.e.m. DAPI, 4,6-diamidino-2-phenylindole; NS, not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/26081042), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Neuropilin-1 by Western Blot Expression of candidate genes. (A) qPCR analysis of relative mRNA levels in total RNA from isolated glomeruli of Foxc2fl/fl; Pod‐Cre+, Foxc2fl/+; Pod‐Cre+ and Foxc2fl/+ mice. Expression levels were normalized against the expression of Rplp0. (B) qPCR analysis of relative mRNA levels in total RNA from a podocyte cell line treated with either Foxc2 siRNA or non‐targeting siRNA. (C) Western blot analysis of NRP1 and ITGB1 protein expression after Foxc2 induction (pBABE‐Foxc2) or knockdown (Foxc2 siRNA). Empty vector (pBABE) and non‐targeting siRNA was used as controls. *P < 0.05 Foxc2fl/fl;Pod‐Cre+ versus Foxc2fl/+. Error bars in (A) and (B) represent SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/31062503), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Neuropilin-1 by Western Blot Nrp1 influences Smad2/3 activation.(a–c) Representative confocal images of wt sprouts from EBs immunolabelled for pSmad2. The outline of the wt sprouts is indicated with a dashed line using the endogenous DsRED marker. DAPI staining (not shown) was used to mark nuclei (blue line). Sprouts from (a) untreated EBs, (b) treated with 2 ng ml−1 Tgf-beta for 1 h and(c) treated with 10 μM SB-421543 for 4 h. Scale bar, 13 μm. (d,e) Western blot analysis of proteins from P4 HUVEC transfected with control siRNA and NRP1 siRNA, with or without stimulation with 2 ng ml−1 TGF-beta for 1 h. Full western blots are shown in Supplementary Fig. 10. A representative blot of six is shown; P=0.0012 NRP1 siRNA compared with control. (f,g) Proteins from P4 HUVEC transfected with control-GFP and NRP1–GFP–His construct for 24 h, with or without stimulation with 2 ng ml−1 TGF-beta for 1 h were assessed for SMAD2 phosphorylation. Full western blots are shown in Supplementary Fig. 11. A representative blot of four is shown; P=0.0017 NRP1 overexpression compared with control. (e,g) Quantification of pSMAD2 protein normalized to SMAD2/3. (h–k) Western blot analysis of proteins from P4 HUVEC transfected with control siRNA and NRP1 siRNA, with or without stimulation with 2 ng ml−1 TGF-beta for 1 h. Full western blots are shown in Supplementary Figs 12 and 13. (i) Quantification of pSMAD2 protein normalized to SMAD2/3; P=0.0848 Nrp1 siRNA compared with control. (k) Quantification of pSMAD3 protein normalized to SMAD3; P=0.0181 NRP1 siRNA compared with control. (l,m) Western blot analysis of P4 HUVEC transfected with control siRNA and NRP1 siRNA, with or without stimulation with 10 ng ml−1 BMP9 for 15 and 30 min. Full western blots are shown in Supplementary Fig. 14. A representative blot of four is shown. (m) Quantification of pSMAD2/3 protein normalized to SMAD2; P<0.0011 NRP1 siRNA compared with control. All values represent mean±s.e.m. DAPI, 4,6-diamidino-2-phenylindole; NS, not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/26081042), licensed under a CC-BY license. Not internally tested by R&D Systems.