Human Neuropilin-1 Antibody

Detection of Human Neuropilin‑1 by Western Blot. Western blot shows lysates of MDA‑MB‑231 human breast cancer cell line and human placenta tissue. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human Neuropilin‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3870) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for Neuropilin‑1 at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Neuropilin‑1 in HUVEC Human Cells by Flow Cytometry. HUVEC human umbilical vein endothelial cells were stained with Sheep Anti-Human Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3870, filled histogram) or control antibody (Catalog # 5-001-A, open histogram), followed by NorthernLights™ 637-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # NL011).

Neuropilin‑1 in PC-3 and HLDM-2 Human Cell Lines. Neuropilin-1 was detected in immersion fixed PC-3 human prostate cancer cell line (positive staining) and HLDM-2 human Hodgkin's lymphoma cell line (negative staining) using Sheep Anti-Human Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3870) at 5 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Neuropilin-1 in Human Pancreatic Cancer Tissue. Neuropilin-1 was detected in immersion fixed paraffin-embedded sections of human pancreatic cancer tissue using Sheep Anti-Human Neuropilin-1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3870) at 10 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-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific labeling was localized to the cytoplasm and plasma membrane of cancer cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human Neuropilin-1 by Knockdown Validated NRP1-dependent EGFR/ERK signalling pathways are activated by EBV and are also associated with the promotion of EBV infection.(a,b) EBV infection activated EGFR/AKT and EGFR/ERK pathways. Serum-starved HNE1 cells were infected with EBV (a) or purified EBV (b) at an MOI of 5 × 103 for the indicated times. (c) EBV-activated EGFR/AKT or EGFR/ERK pathways were partially mediated by NRP1. After transfected with the indicated siRNA duplexes for 48 h, HNE1 cells were serum-starved and infected with EBV at an MOI of 5 × 103 for 1 h, followed by western blotting. For a–c, the intensity of the bands, determined with the ImageJ software, was shown as indicated. alpha -Tubulin, internal control. (d) Schematic diagram of RTKs (EGFR and VEGFR2)/NRP1/Ras/ERK and RTKs/NRP1/PI3K/AKT signalling cascade inhibitors targeting the indicated members of the pathways are shown. (e) EGFR/Ras/ERK signalling pathway was required for EBV infection. EGF-treated HNE1 and NPEC1-Bmi1 cells pre-incubated with Gefitinib (20 μM), Sorafenib (HNE1, 20 μM; NPEC1-Bmi1, 5 μM), U0126 (50 μM) and LY294002 (50 μM) for 30 min were infected with EBV for 2 h, and then cultured in the fresh medium for 48 h until FACS analysis for the infection efficiency. (f,g) Downregulation of EGFR and c-Met impaired EBV infection. HNE1 cells were transfected with siRNA duplexes targeting EGFR or c-Met for 48 h, followed by real-time PCR analysis for the expressions of EGFR and c-Met (f) or FACS analysis for EBV infection efficiency (g). (h) HRas partially rescued the inhibitory effect of Gefitinib on EBV infection. HNE1 cells transfected with pMSCV-HRAS-V12 plasmid for 24 h were pre-incubated with 20 μM Gefitinib for 30 min, followed by exposure to EBV. (i) HRas failed to rescue the suppressive effect of silencing NRP1 on EBV infection. After transfected with siNRP1 or siControl for 36 h, HNE1 cells were reseeded, and then transfected with the plasmid for HRAS-V12 or the empty vector for 24 h, followed by exposure to EBV. The infected cells were analysed by FACS, with controls set to 100%. For (e–i), data represent three times independent experiments. Graphs show mean±s.e.m. ***P<0.001; **P<0.01; *P<0.05; Student’s t-test. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25670642), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Neuropilin-1 by Knockdown Validated NRP1 enhances EBV infection, while NRP2 suppresses EBV infection.(a,b) Downregulation of NRP1 impaired, whereas knockdown of NRP2 promoted EBV infection. HNE1 cells were transfected with siRNA duplexes targeting NRP1 or NRP2 for 48 h, followed by NRP expression analysis by real-time PCR (a) or analysis for the efficiency of EBV infection (b); n=3. (c) EBV infection was blocked by soluble NRP1ABC, but enhanced by antibody against NRP2. For NRP1ABC protein-blocking experiment, HNE1 cells were infected with EBV, which was pre-incubated with purified NRP1ABC for 1 h. For antibody against NRP2-blocking experiment, HNE1 cells were pre-incubated with an anti-NRP2 antibody (100 μg ml−1) or goat IgG (control) at 4 °C for 1 h and then were exposed to EBV at an MOI of 5 × 103 for 3 h at 4 °C. (d,e) Overexpression of NRP1 enhanced EBV infection, while NRP2 suppressed EBV infection. HNE1 cells were transiently transfected with the expression plasmid for NRP1, NRP2 or the empty vector (pMSCV) for 24 h, followed by analysis for the expression of NRP1 and NRP2 by western blotting (d) or were exposed to EBV (e). (f,g) EGF upregulated NRP1 expression and enhanced EBV infection. HNE1 cells cultured with 10 ng ml−1 EGF for 24 h were analysed for the expression of NRP1 and NRP2 by western blotting (f) or were exposed to EBV (g). (h,i) EGF-enhanced EBV infection was partially dependent on NRP1. After transfected with siRNA against NRP1 for 48 h, EGF-treated HNE1 and NPEC-Bmi1 cells maintained in KSF medium supplemented with EGF were analysed for NRP1 expression by western blotting (h) or were exposed to EBV (i). For (b), (c), (e), (g) and (i), HNE1 or NPEC1-Bmi1 cells were exposed to EBV at an MOI of 2.5 × 103 for 2 h at 37 °C, unless otherwise indicated. The percentage of GFP-positive infected cells was analysed by FACS 48 h post infection, with controls (empty vector-transfected cells or vehicle treated-cells) set to 100%. Data represent three to five independent experiments. Values in all graphs are means±s.e.m. ***P<0.001; **P<0.01; *P<0.05; Student’s t-test. For (d), (f) and (h), GAPDH served as an internal control. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25670642), licensed under a CC-BY license. Not internally tested by R&D Systems.