Human VEGF₁₆₅ Antibody

Detection of Human VEGF by Western Blot. Western blot shows lysates of human lung tissue, human kidney tissue, and human liver tissue. PVDF membrane was probed with 5-10 µg/mL of Goat Anti-Human VEGF ₁₆₅Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-293-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). A specific band was detected for VEGF at approximately 27 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

VEGF in HUVEC Cells. VEGF was detected in immersion fixed human umbilical vein endothelial cells (HUVECs) using Goat Anti-Human VEGF₁₆₅ Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-293-NA) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (yellow; NL001) and counter-stained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

VEGF in Human Liver Cancer Tissue. VEGF was detected in immersion fixed paraffin-embedded sections of human liver cancer tissue using Goat Anti-Human VEGF₁₆₅ Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-293-NA) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in cancer cells. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Cell Proliferation Induced by VEGF₁₆₅and Neutralization by Human VEGF Antibody. Recombinant Human VEGF₁₆₅ (293-VE) stimulates proliferation in HUVEC human umbilical vein endothelial cells in a dose-dependent manner (orange line). Proliferation elicited by Recombinant Human VEGF₁₆₅ (10 ng/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Human VEGF₁₆₅ Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-293-NA). The ND₅₀ is typically 0.02‑0.12 µg/mL.

Detection of Human VEGF by Western Blot VEGF expression determined by Western blot and immunoprecipitation.A. Western blot using LiCor Odyssey to simultaneously image pan-VEGF and VEGF-A165b probed western blot. Two different podocyte samples, and a primary RPE sample were run on a gel and probed with antibodies to VEGF-A165b (mouse monoclonal anti-CTRSLTRKD, and 680nm-donkey anti-mouse, top image) and pan-VEGF (rabbit polyclonal anti-VEGF, and 800nm-donkey anti-rabbit, middle image). The bottom image is the pseudocoloured combined image (600nm green, 800nm red). Note the red VEGF165, but yellow VEGF-A165b. MWM = molecular weight marker. d = dimer, m = monomer. B. Protein extracted from human cell lines (adenoma and adenocarcinoma(AC)) subjected to immunoprecipitation (IP) for VEGF-A165b and immunoblotting (IB) for total VEGF-A. A clear strong band was seen in the IP for both cell types at ∼23kDa and ∼46kDa, consistent with the IP for recombinant human VEGF-A165b. A weaker band was seen in the input protein (not subjected to IP), and a second band slightly higher in the AC. A weak band at approximately 56kDa and 28kDa was seen in all lanes subjected to IP, including the VEGF-A165a band, but not seen in the recombinant human VEGF-A165b not subjected to IP, indicating that this is cross reactivity with the IgG. This band was clearly above the VEGF-A165b bands. C. Protein extracted from human cell lines (adenoma and adenocarcinoma(AC)) subjected to immunoprecipitation (IP) for VEGF-A and immunoblotting (IB) for VEGF-A165b. A clear strong band was seen in the IP for both cell types at ∼23kDa, the same size as recombinant human VEGF-A165b. In the input a band at ∼46Da was seen predominantly, for both cell types, labelled as VEGF-A165b dimers. D. Mouse tissues probed with VEGF-A165b antibody detect mouse IgG due to the secondary antibody. Top image, western blot of mouse tissues, recombinant mouse IgG or human VEGF-A165b or VEGF-A165b probed with mouse anti-CTRSLTRKD, and 680nm-donkey anti-mouse IgG. Bottom image blot of same tissues, probed without primary antibody. The same bands are seen in the mouse tissues. Spl = spleen, Col = colon, Hrt = heart, Lng = lung, Liv = liver, Kid = kidney. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23935865), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human VEGF by Western Blot Inhibitor of differentiation 4 (ID4) expression in breast cancer cells leads to the activation of an angiogenic programme in macrophages. a Expression matrix representing a panel of angiogenic factors evaluated using TaqMan Low-Density Arrays (TLDA) in macrophages obtained by 1,25-dihydroxyvitamin D3 (VitD3)-mediated differentiation of HL60 cells and subsequently cultured in RPMI medium or in conditioned media (CM) from control (EV) or ID4-overexpressing (ID4) MDA-MB-468 breast cancer cells. b Western blot showing ID4-HA overexpression in MDA-MB-468 cells. c Selected genes modulated in the arrays were evaluated by RT-qPCR in macrophages obtained from VitD3-mediated differentiation of U937 cells and subsequently cultivated in RPMI medium (CTR) or in CM from control (CM si-SCR) or ID4-depleted (CM si-ID4) MDA-MB-468 cells. d Western blot analysis showing the level of ID4 protein after transfection of the indicated small interfering RNAs (siRNAs) in MDA-MB-468 cells. e–g Western blot analysis of ephrin B2 (EphB2), granulin (GRN) and hypoxia-inducible factor (HIF)-1A proteins in differentiated U937 cells cultured in CM si-SCR or CM si-ID4 from MDA-MB-468 cells. h Immunofluorescence analysis of HIF-1A protein performed in differentiated U937 cells cultured in the presence of CM si-SCR or CM si-ID4 from MDA-MB-468 cells. i Western blotting showing the efficiency of vascular endothelial growth factor A (VEGFA) depletion by siRNA transfection in MDA-MB-468 cells used to prepare CM used in experiments shown in (j). j RT-qPCR analysis of the indicated messenger RNAs in U937 macrophages cultivated in the presence of CM from control (si-SCR) or VEGFA-depleted (si-VEGFA) MDA-MB-468 cells. k RT-qPCR analysis of the indicated genes in differentiated U937 cells cultivated in RPMI medium or in CM from MDA-MB-468 cells in the presence of VEGFA blocking antibody (Ab) or a control Ab. Specifically, VEGFA blocking Ab or control Ab were incubated with CM for 30 minutes at room temperature and CM plus Ab was subsequently used to culture U937 cells for 48 hours. Results from at least three biological replicates are shown. Data are presented as mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005 calculated by two-tailed t test. Image collected and cropped by CiteAb from the following open publication (https://breast-cancer-research.biomedcentral.com/articles/10.1186/s13058-018-0990-2), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human VEGF by Western Blot Western blot analysis of the effects of RNA interference on VEGF expression in HTF cells. (a) Relative expression of VEGF in cells treated with shRNA-2 and shRNA-3; (b) protein expression levels in treated groups and naïve HTF controls. ∗∗∗p < 0.01. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28168047), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of VEGF in Human Liver. Formalin-fixed paraffin-embedded tissue sections of human liver were probed for VEGF mRNA (ACD RNAScope Probe, catalog #423168, Fast Red chromogen, ACD catalog # 322750). Adjacent tissue section was processed for immunohistochemistry using goat anti-human VEGF polyclonal antibody (R&D Systems catalog # AF-293-NA) at 5ug/mL with overnight incubation at 4 degrees Celsius followed by incubation with anti-goat IgG VisUCyte HRP Polymer Antibody (Catalog # VC004) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue). Specific staining was localized to hepatocytes.