The HepG2/HUVEC cells were divided into five groups, including a control, a negative inhibitor (NC) control, a NEAT1 inhibitor, a mimic NC, and a NEAT1 mimic group, as well as a miR-125a-5p treatment group. HepG2 cells and HUVEC were transfected with 100 nM NEAT1/miR-125a-5p mimic, mimic NC, NEAT1/miR-125a-5p inhibitor, or NC inhibitor using Lipofectamine 3000 for 48 h. The transfection efficacy was evaluated by qRT-PCR. The cells in the VEGF group were treated with 50 ng/mL VEGF (cat#ab9571, Abcam) for 24 h.
Vascular endothelial growth factor (vegf)
Manufactured by Abcam
Sourced in United States, United Kingdom, China
VEGF (Vascular Endothelial Growth Factor) is a key regulator of angiogenesis, the process of new blood vessel formation. It is a secreted protein that stimulates the growth and proliferation of endothelial cells, which are the cells that line the interior of blood vessels. VEGF plays a crucial role in various physiological and pathological processes, including embryonic development, wound healing, and the formation of new blood vessels in tumors.
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Lab products found in correlation
Mmp 9, by Abcam (21 mentions)
β actin, by Abcam (20 mentions)
Hif 1α, by Abcam (20 mentions)
Gapdh, by Abcam (17 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (17 mentions)
Pvdf membrane, by Merck Group (14 mentions)
Gapdh, by Cell Signaling Technology (13 mentions)
β actin, by Santa Cruz Biotechnology (12 mentions)
Bcl 2, by Abcam (11 mentions)
P akt, by Cell Signaling Technology (11 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (11 mentions)
Bcl 2, by Cell Signaling Technology (11 mentions)
Cleaved caspase 3, by Cell Signaling Technology (9 mentions)
β catenin, by Abcam (8 mentions)
Gapdh, by Santa Cruz Biotechnology (8 mentions)
Basic fibroblast growth factor (bfgf), by Abcam (8 mentions)
β actin, by Cell Signaling Technology (8 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (8 mentions)
Runx2, by Abcam (7 mentions)
Caspase 3, by Abcam (7 mentions)
269 protocols using vascular endothelial growth factor (vegf)
1
NEAT1 and miR-125a-5p regulation in HepG2 and HUVEC
2
Kidney Tissue Characterization Protocol
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Biopsies of de‐ and recellularized kidneys were fixed overnight in 4% formaldehyde (Klinipath, Duiven, the Netherlands), stored in 70% ethanol and embedded in paraffin. Four‐micrometer‐thick sections were cut and following rehydration of the tissue samples, antigen retrieval was performed by heating the slides in citrate buffer (pH 6). Primary antibodies against laminin (Sigma‐Aldrich, Zwijndrecht, the Netherlands), collagenIV (NovusBio, Abingdon, UK), fibronectin (Abcam, Cambridge, UK), CD31 (Pharmingen), VEGF (Biovision, Zutphen, the Netherlands), and angiopoietin‐1 (Santa‐Cruz, Huissen, The Netherlands), were used. GAGs were shown with the lectins WGA (Invitrogen, Carlsbad, CA), LEA (Sigma‐Aldrich), heparan sulfate specific antibodies (clone 10E4 [Amsbio]), and JM403 (Dr. J. van der Vlag, RadboudUMC, Nijmegen), and a fluorescent‐labeled hyaluronan‐specific binding protein neurocan‐GFP (custom made, adapted from previous published protocol16 ). As secondary antibodies, anti‐mouse, anti‐rat, and anti‐rabbit Alexa Fluor 488, 568, and 647 (Molecular Probes) were used. Nuclei were counterstained using Hoechst 33258 (ThermoFisher). Sections were imaged using SP8‐WLL confocal microscopy and displayed using LAS‐X software (Leica).
3
Quantifying VEGF and HIF-1α Levels
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Both VEGF and HIF-1α protein levels were quantitated via ELISA assay (Biovision, VEGF cat# K5363, HIF-1α cat#: E4285). Briefly, cell lysates were obtained by using lysis buffer, then 50 µL cell lysates were transferred into antibody-coated 96 well plates. Then, streptavidin-HRP solution (50 μl) and 10 μl antibody (VEGF or HIF-1α) were added to each well. Then the sealing membrane was sealed and plates were incubated 60 minutes at 37 ℃. After two washing steps with washing buffer, seconder antibodies were added to all wells and incubated at 37ºC for 1 h. Colorimetric reading was performed by ELISA reader at 450 nm (with 650 nm reference wavelength). Concentrations of both VEGF and HIF-1α were calculated from the standard curves. The minimum detection limit of HIF-1α was 1 pg/mL and VEGF was 20 ng/L.
4
Molecular Profiling of CRC Cells
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Western blotting was performed as previously described17 . In brief, the whole-cell or nuclear extracts from CRC cells and tumor samples were prepared according to a standard protocol. Then, the samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation. Afterwards, the separated samples were transferred onto the polyvinylidene difluoride (PVDF) membranes (Millipore, CA). Later, the membranes were blocked with 5% milk and incubated with the following primary antibodies, including p65 and p-p65 (Santa Cruz Biotechnology, CA, USA), cyclin D1, VEGF, Bcl-2, Survivin, Ki67 (Abcam, Cambridge, UK), cleaved Caspase-3, p-IKK-β, and p-IKK−α (Cell Signaling, MA, USA) antibodies, at 4 °C overnight, followed by incubation with secondary antibodies. Finally, the proteins were visualized using the ECL detection reagent.
5
Comprehensive Western Blot Analysis
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6
Immunohistochemical Analysis of Periodontal Tissues
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Rat periodontal tissues were fixed in 4% PFA for 48 h and decalcified with 12% ethylenediaminetetraacetic acid (EDTA, pH 5.6) at room temperature for 21 days. The sections were paraffin embedded and cut serially in the sagittal plane from the most lingual side. Semi-serial 5-μm sections of first molars were prepared and stained according to immunohistochemistry protocols. After antigen retrieval by heat-induced epitope retrieval, deparaffinized sections were immersed in 0.6% H2O2 for 20 min to quench endogenous peroxidase activity. Sections were then blocked in 5% BSA for 30 min and incubated with antibodies against HIF-1α (0.05 μg/ml), VEGF (0.05 μg/ml), or RUNX2 (0.05 μg/ml) (all from Abcam) overnight at 4°C. Sections were then incubated with goat anti-rabbit or mouse IgG antibodies for 1 h at room temperature and reacted with avidin-biotin-peroxidase complexes (Vector Laboratories, USA) in PBS for 30 min. After colour development with 0.05% 3,3′-diaminobenzidine, sections were counterstained with haematoxylin.
7
Immunohistochemical Analysis of Vaginal Tissue
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A total of 10 sections from each group were incubated with antibodies to Von Willebrand Factor (1:100) (Abcam, Cambridge, MA, USA), VEGF (1:100) (Abcam, Cambridge, MA, USA) and MMP-9 (1:200) (Cell Signaling, Beverly, MA, USA) overnight at 4 degrees Celsius. Slides were rinsed in PBS, followed by treatment using the second antibody using the Max Vision HRP-Polymer anti-Rabbit IHC Kit, (Maxim Co. China). The morphological changes of vaginal tissue were observed under the microscope and photographed (German Leica binocular microscope DM500, × 400). The vaginal tissue in each group is 10, that is, (n = 10). When P < .05, the difference was statistically significant.
8
Protein Expression Analysis of Anterior Vaginal Tissue
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Anterior vagina tissue protein samples were prepared by homogenizing in RIPA lysis buffer. Equal protein (20 μg/lane) was electrophoresed on 10% SDS-PAGE and then transferred to polyvinylidene fluoride membrane (Millipore Corp, Bedford, MA, USA). Western blot was performed with antibodies against MMP-9 (1:400) (Cell Signaling, Beverly, MA, USA), VEGF (1:800) (Abcam, Cambridge, MA, USA), SMA (1:1000) (Santa Cruz Biotechnology, CA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000) (Santa Cruz Biotechnology, CA, USA).The tissue from each animal kept separate, and 1 sample from each treatment group was run on each gel, so 4 lanes were run per gel. More than 3 times were this repeated on different gels.
9
Western Blot Analysis of Cellular Proteins
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Total protein was extracted with RIPA buffer and protein concentration was determined using the DC protein Assay Kit I (Bio-Rad, Hercules, CA, USA). All protein samples were denatured at 100 °C for 5 min. An equivalent amount of the protein was isolated using SDS-PAGE and subsequently transferred to a PVDF membrane (Amersham, Arlington, IL, USA). The membrane was immersed in 5% fat-free milk powder, blocked the heterologous antigens at room temperature for 1 h, and then incubated the major antibodies overnight at 4 °C. After washing with PBST three times, membranes were incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Signals were detected and recorded using an ECL kit (Millipore, Bedford, MA, USA) and a polychromatic fluorescence chemiluminescence imaging analysis system (Alpha, San Leandro, CA, USA), and quantified using densitometry (Image J version 1.8.0, National Institutes of Health, Bethesda, MD, USA), respectively. Antibodies used in Western blot included Nrf2, Keap1, GAPDH, MDM2, VEGF, HO-1 (Abcam, Cambridge, MA, USA), p62, and Bcl-2 (Santa Cruz Biotechnology, Piscataway, NJ, USA).
10
Western Blot Analysis of Signaling Proteins
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Sample extracts (30μg protein) were resolved by SDS-PAGE and transferred to nitrocellulose membranes, blocked with 5% skim milk and probed for monoclonal GSK3β and LRP5 (Abcam), LEF1 (Millipore)or polyclonal pY216 GSK3β, VEGF (Abcam) primary antibodies at a concentration of 1μg/ml. Membranes were then washed and blotted with the appropriate secondary antibody (Dako). Band densities were determined with the ChemiDoc XRS system (Bio-Rad) in chemiluminescence detection modus and Quantity-One software (Bio-Rad). Normalization was performed against β-ACTIN.
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