Cells infected with the indicated lentiviral constructs were lysed in RIPA buffer (Solarbio, Beijing, China) with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, Sigma-Aldrich Inc., Milwaukee, Wisconsin, USA) for 20 minutes at 4°C. After centrifugation, protein concentrations were measured using the BCA Protein Assay Kit (TIANGEN Biotech, Beijing, China). An amount of 50 µg protein from each sample was subjected to SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Immobilon P, Merck Millipore Corporation, Billerica, MA, USA). The membrane was cut into sections based on protein markers, and then probed for Sema4D, Plexin-B1, and VEGF, with α-tubulin selected as a loading control. The antibodies used were as follows: mouse anti-Sema4D (610670, BD Biosciences); mouse anti-Plexin-B1 (sc28372, Santa Cruz A8); mouse anti-VEGF (sc74584, Santa Cruz Biotechnology Inc., Dallas, TX, USA); and VEGFR2 (9698, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-α-tubulin (Epitomics, Inc., Burlingame, CA, USA). The membranes were then incubated with the appropriate secondary antibodies (goat anti-mouse IgG [Heavy and Light Chain] peroxidase labeled [KPL], goat anti-rabbit IgG [Heavy and Light Chain] peroxidase labeled, KPL, Gaithersburg, MD, USA). Proteins were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore).
Mouse anti vegf
Manufactured by Santa Cruz Biotechnology
Sourced in United States
This Mouse anti-VEGF product is a monoclonal antibody that specifically binds to the Vascular Endothelial Growth Factor (VEGF) protein. It can be used for the detection and quantification of VEGF in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti flk 1, by Santa Cruz Biotechnology (3 mentions)
Mouse anti rptpβ ζ, by BD (2 mentions)
Goat anti rptpβ ζ, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Mouse anti sema4d, by BD (1 mentions)
Vegfr2, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Tiangen Biotech (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti yap, by Cell Signaling Technology (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Glass slides, by Ibidi (1 mentions)
Mouse anti ncl, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti flk1, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti vegf, by Santa Cruz Biotechnology (1 mentions)
Mouse anti ανβ3, by Merck Group (1 mentions)
Quantifast sybr green pcr kit, by Qiagen (1 mentions)
9 protocols using mouse anti vegf
1
Western Blot Analysis of Semaphorin Signaling
2
EGCG Modulation of VEGF in 3T3-L1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3T3-L1 cells (5 × 105 cells/well) were cultured in 12-well plates. After confluent, 3T3-L1 cells treated with differentiation medium and different concentrations of EGCG (0, 5, 10, and 25 µg/mL) for 8 days. Then, 3T3-L1 cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.2% Triton X-100 for 20 min at room temperature. Cells were washed 3× with 300 µL PBS and incubated with a primary antibody against VEGF (mouse anti-VEGF, Santa Cruz, Cambridge, UK, dilution 1:200) for 2 h in a humidified chamber. After three further washes with PBS, cells were incubated with Alexa Fluor®488 labeled anti-mouse antibody (Sigma–Aldrich, St. Louis, MO, USA) for 1 h. Subsequently, cells were washed three times with PBS. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Then, cells were washed with PBS and viewed using a fluorescence microscope.
3
Retinal Injury Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPE-choroid-sclera complexes and retinas were extracted from three mice at 1, 3, 7, and 14 days after laser injury. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was then incubated with rabbit anti-YAP (1:500; Cell Signaling Technology, Danvers, MA), mouse anti-VEGF (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-PCNA (1:1,000; Chemicon, Temecula, CA). The antibodies were incubated with 5% skim milk overnight at 4 °C and reacted with horseradish peroxidase (HRP)–conjugated secondary antibodies (1:2,000; Thermo Scientific, Rockford, IL) at 37 °C for 2 h. Extensive washes in 0.05% Tween-20 in TBS were followed by incubation with anti-GAPDH (1:1,000; Sigma-Aldrich, Saint Louis, MO). The blots were then incubated with chemifluorescent reagent enhanced chemiluminescence (ECL; Thermo Scientific) and exposed to X-ray film in the dark. The intensity of the GAPDH signal was used as an endogenous control, and band optical density was quantified using ImageJ (National Institutes of Health, Bethesda, MD).
4
In situ Proximity Ligation Assay for Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For detection of protein-protein interactions, in situ PLA was performed. The components used (Sigma) were as follows: anti-mouse PLA plus probe, anti-rabbit PLA minus probe and Detection Reagents Orange. HUVEC or U87MG cells were grown on μ-Chamber 12 well on glass slides (Ibidi©, Martinsried, Germany). After reaching 80% confluence or after appropriate treatment of cells, the assay was performed according to the manufacturer’s instructions. Briefly, after fixation and blocking, cells were incubated with the primary antibodies: mouse anti-VEGF (1:250), rabbit anti-VEGF (1:250), rabbit anti-Flk-1 (1:250), mouse anti-Flk-1 (1:250), mouse anti-NCL (1:50), goat anti-RPTPβ/ζ (1:250) (all from Santa Cruz Biotechnology Inc.), mouse anti-ανβ3 (1:500; Merck Millipore), mouse anti-PTN (1:500; Abnova, Heidelberg, Germany) and mouse anti-RPTPβ/ζ (1:250; BD Biosciences). Subsequently, cells were incubated with secondary antibodies conjugated with oligonucleotides. After hybridization and ligation of the oligonucleotides, the DNA was amplified. A detection mixture detected the amplicons, resulting in red fluorescence signals. Nuclei were counterstained with Draq5; cells were mounted with Mowiol 4–88 and visualized at room temperature with Leica SP5 confocal microscope.
5
Protein Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA buffer, as previously described [8 (link)]. Three mg of total protein were incubated with primary antibody for 16 h at 4°C under continuous agitation. The primary antibodies used were: mouse anti-VEGF (3 μg), mouse anti-Flk-1 (3 μg), goat anti-RPTPβ/ζ (3 μg), goat anti-PTN (3 μg) (Santa Cruz Biotechnology Inc.) and goat anti-β3 (1.5 μg; Merck Millipore). Protein A- and protein G-agarose beads (Merck Millipore) were added, samples were further incubated for 2 h at 4°C, and beads with bound proteins were collected by centrifugation (5,000 g for 5 min at 4°C) and washed twice with ice-cold PBS pH 7.4. Immunoprecipitated proteins were resuspended in SDS loading buffer and analyzed by Western blot.
6
Src Inhibition and VEGF Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Src PTK inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), was obtained from Merck Calbiochem Co. (Darmstadt, Germany). The primary antibodies used were rabbit anti-Src and anti-p-Src (pY418) (Epitomics, an Abcam Co. Burlingame, CA, USA), mouse anti-VEGF (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), claudin-5 monoclonal antibody (CLDN5; Invitrogen, Life Technologies, Carlsbad, CA, USA), rabbit anti-glial fibrillary acidic protein (GFAP; Boster, Wuhan, China) and rabbit polyclonal antibody to β-actin (Beijing Biotech Co., Ltd.). The following secondary antibodies were used: Alexa Fluor 594- conjugated AffiniPure goat anti-rabbit IgG, Alexa Fluor 488- conjugated AffiniPure goat anti-mouse IgG [Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. (ZsBio), Beijing, China], goat anti-rabbit or goat anti-mouse secondary antibody (Histostain-Plus kits; ZsBio). A QuantiFast SYBR-Green PCR kit (Qiagen, Hilden, Germany) was used for the quantitative (real-time) polymerase chain reaction (PCR) assays.
7
Immunoblot Analysis of Angiogenic Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were analyzed by SDS-PAGE and transferred to Immobilon P membranes. Blocking was performed by incubating the membranes with Tris-buffered saline (TBS) pH 7.4 with 0.05% Tween (TBS-T), containing either 5% nonfat dry milk or 3% BSA. Membranes were incubated with primary antibodies for 16 h at 4°C under continuous agitation, washed 3 times with TBS-T, and incubated with secondary antibodies for 1 h at room temperature. Primary antibodies used were mouse anti-Flk-1 (1:500), goat anti-β3 (1:500), mouse anti-VEGF (1:500), rabbit anti-phospho-β3(Y773) (1:1,000; Santa Cruz Biotechnology Inc.), mouse anti-RPTPβ/ζ (1:500; BD Biosciences), rabbit anti-c-Src (1:1,000; Merck Millipore), rabbit anti-phospho-c-Src(Y418) (1:1,000; Acris Antibodies GmbH) and rabbit anti-non-phospho-c-Src (1:1,000; Cell Signaling Technology Inc.). Detection of immunoreactive bands was performed using the enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL, USA). Protein levels were quantified using the ImagePC image analysis software (Scion Corp., Frederick, MD, USA).
8
Isolation and Characterization of Endothelial Progenitor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
G-CSF was provided by UBI pharma Inc. (Taipei, Taiwan). Fluorescein isothiocyanate (FITC) anti-mouse Sca-1 and phycoerythrin (PE) anti-mouse Flk-1 antibodies were purchased from eBioscience (San Diego, CA, USA). Isotype-identical antibodies were obtained from Becton Dickinson (Franklin Lakes, NJ, USA). The antibodies listed below were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): rabbit anti-eNOS, anti-p-eNOS, mouse anti-VEGF, anti-α-SMA, and anti-β-actin. Rabbit antibodies for STAT3 and p-STAT3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse antibodies for IL-10 were purchased from Serotec (Oxford, UK). Rat anti-CD31 was obtained from BD Pharmingen (San Diego, CA, USA). Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor, Histopaque-1077 (density: 1.077 g/mL), and 2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Endothelial growth medium (EGM-2 MV) and growth factors for EPC culture were obtained from Lonza (Morristown, NJ, USA).
9
Immunostaining of Retinal Müller Cells
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!