Total proteins were extracted using RIPA lysis buffer within 1% protease inhibitor cocktail (Millipore, Bedford, MA, USA) 0.1 mM according to the manufacturer’s instructions. Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). After blocking, the membranes were then incubated at 4 °C overnight with the following primary antibodies: anti-HIF-1α, anti-VEGF-A, anti-JNK, anti-PPAR-γ, anti-C/EBP-α, anti-FABP4 (1:1000, all Cell Signaling Technology, Danvers, MA, USA), anti-NF-κB p65, anti-p-IκB, anti-TNF-α, anti-MCP-1 (1:1000, all Abcam, Cambridge, MA, USA), anti-SFRP5, anti-Wnt5a, anti-Wnt10b, anti-β-catenin (1:1000), and anti-β-actin (1:5000, all GeneTex, Irvine, CA, USA). After washing, the membranes were probed with corresponding second antibodies (1:3000, GeneTex). The density of the individual protein bands was quantified by densitometric scanning of the blots using ImageJ software.
Anti vegfa
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-VEGFA is a primary antibody that binds to the Vascular Endothelial Growth Factor A (VEGFA) protein. VEGFA is a key regulator of angiogenesis, the process of new blood vessel formation. The Anti-VEGFA antibody can be used in various research applications to detect and study the VEGFA protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Cell Signaling Technology (5 mentions)
Anti p akt, by Cell Signaling Technology (4 mentions)
Anti hif 1α, by Cell Signaling Technology (3 mentions)
Anti mouse and anti rabbit peroxidase linked, by Cell Signaling Technology (3 mentions)
Ecl prime western blotting detection reagent, by Advansta (3 mentions)
Anti akt3, by Cell Signaling Technology (3 mentions)
Anti pi3k, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti β actin, by GeneTex (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti pparγ, by Cell Signaling Technology (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Anti c ebpα, by Cell Signaling Technology (1 mentions)
Anti fabp4, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Abcam (1 mentions)
Anti p iκb, by Abcam (1 mentions)
Anti mcp 1, by Abcam (1 mentions)
Anti wnt5a, by GeneTex (1 mentions)
10 protocols using anti vegfa
1
Western Blot Analysis of Adipogenic Markers
2
Western Blot Analysis of Angiogenic Markers
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This analysis was conducted as previously described.14 Cells were lysed in RIPA buffer to obtain total protein samples and the proteins were separated by SDS‐PAGE in a 10% gel and then electrophoretically transferred to a nitrocellulose membrane (Bio‐Rad, Hercules, CA). The membranes were blocked with 5% non‐fat milk and then incubated with the following primary antibodies: anti‐EYA4, anti‐c‐JUN, anti‐p‐c‐JUN(ser73), anti‐VEGFA and anti‐CD31 (Cell Signalling Technology, Beverly, MA) overnight at 4°C. GAPDH served as a loading control. The membranes were incubated with a horseradish peroxidase–conjugated secondary antibody. The protein bands were detected with the ECL Plus Developing System (Amersham Biosciences; Piscataway, NJ).
3
Western Blot Analysis of Akt3 and PI3K
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Whole-cell lysates and total exosomal proteins were prepared by using RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). 50ug total proteins were electrophoretically separated on 4–12% SDS-acrylamide Gel (Thermo Fisher Scientific). Akt3 and PI3K p110α were examined in the study. Western blot analyses were performed with primary antibodies: anti- β-actin, anti-Akt 3, anti-P-Akt, anti-PI3K, anti-VEGFA (1: 1000, Cell Signaling Technology, USA), and the corresponding secondary antibodies anti-mouse and anti-rabbit peroxidase-linked (1: 10 000; Cell Signaling Technology, USA). The signals were visualized by ECL Prime Western Blotting Detection Reagent (Advansta, USA).
4
Endometrial Carcinoma Protein Expression
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Endometrial carcinoma cells were lysed in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific), and the protein concentration of the cellular lysates was determined by acid protein kit (Thermo Fisher Scientific). The lysates were separated by SDS-PAGE and then electro-transferred onto PVDF membrane (Millipore, Bedford, MA, USA). The membranes were blocked in 5% of BSA, and probed overnight with the primary antibodies: anti-VEGFA (1:2,000, Cell Signaling, Beverly, MA, USA) and anti-β-actin (1:3,000, Cell Signaling). Following incubation with horseradish peroxidase-labeled secondary antibody (1:5,000; Cell Signaling), the immunoreactivities were detected by enhanced chemiluminescence (KeyGen, Nanjin, China).
5
Western Blot Analysis of Signaling Proteins
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WB analysis was performed as described previously [17 (link)]. Generally, total protein was extracted by RIPA Lysis Buffer (Thermo, USA). Protein was measured, separated, and transferred to PVDF membranes (Millipore, USA) after further incubation with primary and secondary antibodies overnight at 4°C. The final protein was visualized by the Super ECL Detection Kit (KeyGEN BioTECH, China). The antibodies used in this study were as follows: anti-HIF-1α (1 : 1000, #36169S, Cell Signaling, USA), anti-EFNA3 (1 : 1000, ab153706, Abcam, USA), anti-AKT (1 : 1000, 4691, Cell Signaling, USA), anti-p-AKT (1 : 1000, 4060, Cell Signaling, USA), anti-VEGFA (1 : 1000, #65373, Cell Signaling, USA), anti-Wnt3a (1 : 1000, #2721, Cell Signaling, USA), anti-β-catenin (1 : 1000, #9562, Cell Signaling, USA), anti-RhoA (1 : 600, #2117, Cell Signaling, USA), and anti-β-actin (1 : 5000, 20536-1-AP, Proteintech, USA).
6
Immunofluorescent Staining of VEGF-A
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Cells were washed with cold‐PBS three times, fixed with 4% formaldehyde for 60 minutes and permeabilized using 0.1% triton‐x for 30 minutes. After blocking with 10% normal goat serum for 1 hour at room temperature, the samples were incubated with primary antibodies (anti‐VEGF‐A; 1:1000; Cell Signaling Technology, Massachusetts, USA) for 1 hour at room temperature and gently washed three times in PBS. Then, the samples were incubated with fluorescein (FITC)‐conjugated secondary antibody (1:1000; Cell Signaling Technology) and stained with DAPI (1 μg/mL, Cell Signaling Technology) before image acquisition using a fluorescence microscope (Olympus, Tokyo, Japan).
7
Western Blot Analysis of Cellular Proteins
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Cytosolic protein lysates and nuclear fractions were prepared using RIPA buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland) or nuclear extraction kit (Abcam) respectively. Their concentrations were measured using the BCA assay kit (Thermo Scientific, Waltham, MA, USA). Proteins were separated on 6–12% sodium dodecyl sulfate-polyacrylamide gels by electrophoresis and transferred onto polyvinylidene fluoride membranes. The blots were incubated with the following primary antibodies: anti-LDHB (1:1000, Cell Signaling, Danvers, MA, USA), anti-PDH (1:1000, Cell Signaling), anti-actin (1:10,000, Cell Signaling), anti-HIF-1α (1:1000, Cell Signaling), anti-VEGF-A (1:1000, Cell Signaling), and anti-lamin B (1:1000, Cell Signaling). Subsequently, secondary antibodies were used to detect specific proteins (1:10,000, Cell Signaling).
8
Protein Expression Analysis in Exosomes
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Whole-cell lysates and total exosomal proteins were prepared by using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). 50ug total proteins were electrophoretically separated on 4-12% SDS-acrylamide Gel (Thermo Fisher Scientific). Akt3 and PI3K p110α were examined in the study. Western blot analyses were performed with primary antibodies: anti-β-actin, anti-Akt 3, anti-P-Akt, anti-PI3K, anti-VEGFA (1: 1000, Cell Signaling Technology, USA), and the corresponding secondary antibodies anti-mouse and anti-rabbit peroxidase-linked (1: 10 000; Cell Signaling Technology, USA). The signals were visualized by ECL Prime Western Blotting Detection Reagent (Advansta, USA).
9
Western Blot Analysis of Akt3 and PI3K
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Whole-cell lysates and total exosomal proteins were prepared by using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). 50ug total proteins were electrophoretically separated on 4-12% SDS-acrylamide Gel (Thermo Fisher Scienti c). Akt3 and PI3K p110α were examined in the study. Western blot analyses were performed with primary antibodies: anti-β-actin, anti-Akt 3, anti-P-Akt, anti-PI3K, anti-VEGFA (1: 1000, Cell Signaling Technology, USA), and the corresponding secondary antibodies anti-mouse and anti-rabbit peroxidase-linked (1: 10 000; Cell Signaling Technology, USA). The signals were visualized by ECL Prime Western Blotting Detection Reagent (Advansta, USA).
10
Glioma Cell Protein Expression Analysis
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Glioma cells were treated with different concentrations (0, 12.5, 25, 50 µM) of compound for 24 hours before being lysed by RIPA containing protease inhibitor.
Lysates were incubated on ice for 20 min prior to centrifugation at 12000 rpm for 20 min at 4℃. After being determined concentrations using a BCA kit (Pierce), 30 µg total protein was separated by SDS-PAGE then transferred to the polyvinylidene fluoride membrane. The primary antibodies used were as follows: anti-KDR (Cell Signaling Technology, #2479), anti-PTEN (Cell Signaling Technology, #9188), anti-MMP-9 (Cell Signaling Technology, #13667), anti-MMP-2 (Cell Signaling Technology, #87809), anti-VEGFA (Cell Signaling Technology, #65373), anti-ID2 (Cell Signaling Technology, #3431), anti-β-actin (Cell Signaling Technology, #4970). The dilutions for the antibodies were as follows: 1:1000 for KDR, 1:1000 for PTEN, 1:1000 for MMP-9, 1:1000 for MMP-2, 1:1000 for VEGFA, 1:1000 for ID2, 1:1000 for β-actin, 1:10000 for Goat anti-Rabbit IgG.
Lysates were incubated on ice for 20 min prior to centrifugation at 12000 rpm for 20 min at 4℃. After being determined concentrations using a BCA kit (Pierce), 30 µg total protein was separated by SDS-PAGE then transferred to the polyvinylidene fluoride membrane. The primary antibodies used were as follows: anti-KDR (Cell Signaling Technology, #2479), anti-PTEN (Cell Signaling Technology, #9188), anti-MMP-9 (Cell Signaling Technology, #13667), anti-MMP-2 (Cell Signaling Technology, #87809), anti-VEGFA (Cell Signaling Technology, #65373), anti-ID2 (Cell Signaling Technology, #3431), anti-β-actin (Cell Signaling Technology, #4970). The dilutions for the antibodies were as follows: 1:1000 for KDR, 1:1000 for PTEN, 1:1000 for MMP-9, 1:1000 for MMP-2, 1:1000 for VEGFA, 1:1000 for ID2, 1:1000 for β-actin, 1:10000 for Goat anti-Rabbit IgG.
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