VEGF Antibody (VG1) - BSA Free

Flow Cytometry of U-937 Cells Stained with VEGF Antibody

An intracellular stain was performed on U-937 cells with NB100-664 (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1.0 ug/mL for 30 minutes at room temperature, followed by Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Dylight 550 (35503, Thermo Fisher).

Western Blot Detection of VEGF in Transfected ADMSCs

After transfection with 50 nmol/L miR-1956 mimic for 3 days, the levels of secreted and cellular VEGF were evaluated using western blotting. Representative images of four independent experiments are shown. Data are shown as mean +/- SD. *, significantly different from control; **, p < 0.01. ADMSCs embedded in Matrigel were implanted subcutaneously on the back of mice. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/31938051/) licensed under a CC-BY license.

Western Blot Analysis of VEGF in Transfected Cell Lines

HUVEC and 293T cells transfected with a plasmid expressing human VEGF165 at 1:1000. Image provided by verified customer review.

Immunocytochemistry/Immunofluorescence Staining of VEGF in U87 Cells

U-87 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.05% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti- NB100-664 at 2 ug/ml overnight at 4C and detected with an anti-mouse Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.

Immunohistochemical Analysis of VEGF in Paraffin Embedded Human Angiosarcoma Tissue

FFPE human angiosarcoma tissue section using VEGF antibody (clone VG1). The endothelial cells of the blood vessels and most of the cancer cells showed strong positivity for VEGF protein.

Flow Cytometry of HUVEC Cells Stained with PerCP Conjugated VEGF Antibody

Analysis of PerCP conjugate of NB100-664. An intracellular stain was performed on HUVEC cells with VEGF (VG1) antibody NB100-664PCP (blue) and a matched isotype control NBP2-27287PCP (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 10 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to PerCP.

Flow Cytometry of U-937 Cells Stained with FITC Conjugated VEGF Antibody

An intracellular stain was performed on U-937 cells with NB100-664F (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 10 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to FITC.

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] -

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] - Regorafenib reduced TNBC cell migration & targeted p-STAT3/VEGF-A signaling.(A) Chemical structure of regorafenib. (B,C), Transwell assay (B) & wound-healing assay (C) were performed in TNBC cell lines after regorafenib treatment for 24 h. *p < 0.05, **p < 0.01. (D) dose-dependent effects of regorafenib on p-STAT3 & VEGF-A proteins were analyzed by western blot. (E) Dose-dependent effects of regorafenib on VEGF-A mRNA were analyzed by qPCR. TNBC cells were exposed to the indicated doses for 24 hours. *p < 0.05, **p < 0.01. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27364975), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] -

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] - SC-78, a derivative of regorafenib, exhibited more potent anti-migratory effects than regorafenib.(A) Chemical structure of SC-78. (B) The protein levels of p-VEGFR2, VEGFR2, p-PDGFR, & PDGFR in EAhy926 treated with various doses of regorafenib or SC-78 were determined by western blot. (C,D), The effect of SC-78 on migration of human TNBC cells was analyzed by Transwell migration assay (C) & wound-healing assay (D). *p < 0.05, **p < 0.01. (E) Human TNBC cells were treated with SC-78 dose dependently for 24 h, & the cell lysates were subjected to western blot assay. (F) MDA-MB-231 cells were transfected with vector control or VEGF-A overexpression plasmid (left), STAT3 overexpression plasmid (middle), scramble or SHP-1 siRNA (right), respectively. After 48 h transfection, cells were treated with SC-78 (2.5 μM) for 24 h & subjected to western blot assay or cells analyzed by Transwell migration assay. *p < 0.05, **p < 0.01. (G) SHP-1 activity assay was performed in MDA-MB-231 & MDA-MB-468 cells treated with SC-78 dose dependently. *p < 0.05, **p < 0.01. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27364975), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] -

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] - Anti-tumor activity of regorafenib & SC-78 in a murine TNBC metastasis model.(A) Nude mice were subcutaneously injected with MDA-MB-468 cells (2 × 106). Mice were treated with vehicle, bevacizumab (125 μg/100 μl, twice a week, IP), regorafenib, or SC-78 (10 mg/kg, everyday, oral) for 43 days (n = 6), & the tumor size (left, bottom) was measured. In vivo protein levels were analyzed (Middle, top). Middle, bottom, the in vivo SHP-1 activity. Right, Representative images of IHC staining (100 × ). *p < 0.05, **p < 0.01. (B) Luciferase-expressing MDA-MB-231 (1 × 106) cells were injected orthotopically into the mammary fat pad of the mice. After two weeks, mice received regorafenib & SC-78, or vehicle orally at 10 mg/kg/every day (n = 5). Tumor growth was monitored by IVIS imaging system at the indicated times. Left, top, visualized by IVIS analysis. Left, bottom, quantification analysis from the IVIS total flux. Right, Representative images of IHC staining (100×). *p < 0.05, **p < 0.01. (C) In vivo bioluminescence images of nude mice injected i.v. with MDA-MB-231/Luc2 cells (1 × 106). After bioluminescence was observed, mice received vehicle or SC-78 orally at 10 mg/kg/every day. left, visualized by IVIS analysis. Right, Kaplan–Meier plot showing animal survival after treatment with vehicle or SC-78 (n = 8). The survival endpoint was set at 64 days after drug administration. (D) Schematic displays the drug mechanism of regorafenib & SC-78 on VEGF-A autocrine & paracrine inhibition & cell migration. SC-78 suppressed cancer metastasis dominantly through SHP-1 dependent-STAT3 dephosphorylation. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27364975), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] -

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] - Regorafenib transcriptionally inhibited VEGF-A expression through decreasing binding of STAT3 on promoter of VEGF-A.(A) Top, MDA-MB-231 cells co-transfected w/ a Renilla control vector & plasmids containing firefly luciferase gene driven by wild-type or STAT3 binding site-mutated VEGF-A promoter. After transfection for 48 h, cells treated w/ regorafenib for 24 h. Promoter activity analyzed by luciferase assay after regorafenib treatment. Bottom, MDA-MB-231 cells transfected w/ vector-control or STAT3-overexpression plasmid for 48 h. After , cells further co-transfected w/ Renilla & wild-type VEGF-A promoter & detect promoter activity as mentioned above. *p < 0.05, **p < 0.01. (B) After regorafenib treatment for 24 h, STAT3 binding site fragment detected by PCR in ChIP samples precipitated w/ STAT3 & rabbit IgG control antibodies in MDA-MB-231 cells. (C,D), MDA-MB-231 cells transfected, respectively, w/ control vector or VEGF-A overexpression plasmid (C) or STAT3 overexpression plasmid (D) for 48 h. After transfection, cells treated w/wo regorafenib for 24 h & subjected to WB assay or seeded to Transwell to analyze migration ability. *p < 0.05, **p < 0.01. (E) Cells treated w/ regorafenib at indicated dosages for 24 h & cell lysates analyzed by SHP-1 phosphatase activity assay. *p < 0.05, **p < 0.01. (F) MDA-MB-231 cells pretreated w/ pan-phosphatase inhibitor (left), or specific SHP-1 inhibitor (Middle) for 1 h before regorafenib treatment. Right, MDA-MB-231 cells transfected, respectively, w/ control siRNA or SHP-1 siRNA for 48 h. After transfection, cells treated w/wo regorafenib (10 μM) for 24 h. The protein levels analyzed by WB assay or cells seeded to Transwell to analyze migration ability. *p < 0.05, **p < 0.01. Image collected & cropped by CiteAb from following publication (https://pubmed.ncbi.nlm.nih.gov/27364975), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] -

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] - Notch-1 is identified as an intermediate molecule in the miR-1956-VEGF axis. (A) Notch-1 is a target gene for miR-1956. The sequence of miR-1956, the potential binding site at the 3' UTR of Notch-1 mRNA, & the nucleotides mutated in the Notch-1-3' UTR mutant are shown. HEK293 cells were transiently cotransfected with miR-1956 mimic or NC using luciferase reporter vectors. The luciferase activity was normalized to the activity of Renilla luciferase. Data are shown as mean ± SD from three independent experiments. *, significantly different from NC; **, p < 0.01. After transfection with 50 nmol/L of miR-1956 mimic for 3 days, the mRNA level of Notch-1 was determined using qRT-PCR (B), & the protein expression of Notch-1 was measured using western blotting (C). For B, data are shown as mean ± SD from three independent experiments. *, significantly different from control; **, p < 0.01. For C, representative images of two independent experiments are shown. Data are shown as mean ± SD. *, significantly different from control; **, p < 0.01. ADMSCs were transfected with 2.5 μg Notch-1 plasmid & 50 nmol/L miR-1956 mimic or 100 nmol/L miR-1956 inhibitor for 3 days, the intracellular expression of Notch-1 (D) & VEGF (E) was detected by western blotting. For D & E, representative images of four independent experiments are shown. Data are shown as mean ± SD. *, significantly different from the control group transfected with control plasmid & NC mimic or inhibitor; *, p < 0.01; **, p < 0.01. #, significantly different from the group transfected with Notch-1 plasmid & NC mimic or inhibitor; #, p < 0.05. One-way ANOVA followed by Tukey's post-test was performed (A-E). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31938051), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] -

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] - SHP-1 is negatively associated with p-STAT3/VEGF-A signaling & metastasis in TNBC cells & clinical samples.(A) Protein expression pattern of SHP-1, p-STAT3, & VEGF-A in nine TNBC cell lines were analyzed by western blot. (B) The migration abilities of nine TNBC cell lines were analyzed by Transwell assay. DAPI stains the nuclei. BT-20 cells were used as a normalization control. (C) The correlation (linear regression model) of VEGF-A (top), p-STAT3 (middle), & SHP-1 (bottom) & migration ability in nine TNBC cell lines. (D) TNBC cells from representative two patients with SHP-1, p-STAT3, & VEGF-A staining. (200×) (E) Kaplan-Meier graph was prepared to compare DMFS (left) & DFS (right) in patients with high VEGF-A (H score > 160) or low VEGF-A (H score < = 160) levels for the indicated time of follow up. Chi-square test indicated a significant difference between VEGF-A high (N = 21) & low (N = 76) patients. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27364975), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] -

Western Blot: VEGF Antibody (VG1) - BSA Free [NB100-664] - Regorafenib transcriptionally inhibited VEGF-A expression through decreasing binding of STAT3 on promoter of VEGF-A.(A) Top, MDA-MB-231 cells co-transfected w/ a Renilla control vector & plasmids containing firefly luciferase gene driven by wild-type or STAT3 binding site-mutated VEGF-A promoter. After transfection for 48 h, cells treated w/ regorafenib for 24 h. Promoter activity analyzed by luciferase assay after regorafenib treatment. Bottom, MDA-MB-231 cells transfected w/ vector-control or STAT3-overexpression plasmid for 48 h. After , cells further co-transfected w/ Renilla & wild-type VEGF-A promoter & detect promoter activity as mentioned above. *p < 0.05, **p < 0.01. (B) After regorafenib treatment for 24 h, STAT3 binding site fragment detected by PCR in ChIP samples precipitated w/ STAT3 & rabbit IgG control antibodies in MDA-MB-231 cells. (C,D), MDA-MB-231 cells transfected, respectively, w/ control vector or VEGF-A overexpression plasmid (C) or STAT3 overexpression plasmid (D) for 48 h. After transfection, cells treated w/wo regorafenib for 24 h & subjected to WB assay or seeded to Transwell to analyze migration ability. *p < 0.05, **p < 0.01. (E) Cells treated w/ regorafenib at indicated dosages for 24 h & cell lysates analyzed by SHP-1 phosphatase activity assay. *p < 0.05, **p < 0.01. (F) MDA-MB-231 cells pretreated w/ pan-phosphatase inhibitor (left), or specific SHP-1 inhibitor (Middle) for 1 h before regorafenib treatment. Right, MDA-MB-231 cells transfected, respectively, w/ control siRNA or SHP-1 siRNA for 48 h. After transfection, cells treated w/wo regorafenib (10 μM) for 24 h. The protein levels analyzed by WB assay or cells seeded to Transwell to analyze migration ability. *p < 0.05, **p < 0.01. Image collected & cropped by CiteAb from following publication (https://pubmed.ncbi.nlm.nih.gov/27364975), licensed under a CC-BY license. Not internally tested by Novus Biologicals.