Cells and tissues were lysed by RIPA buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes (Pierce, Rockford, IL, USA). The membranes were blocked with 5% nonfat dry milk in TBST. Primary antibodies were: SMAD7 (MAB2029, R&D Systems, Minneapolis, MN, USA), P21 (SAB4500065, Sigma Aldrich), pSMAD2 (Ser465/467), pSMAD1/3, BCL-2, BCL-XL, pSTAT3, pJNK, pc-JUN, MCL-1, cleaved caspase-3 and Actin (3101, 9520,2870, 2762, 9145, 4668, 3270,5453,9661 and 3700, Cell Signaling Technology), c-MYC, pIκBα, IL-6, TGF-β1, pERK, CyclinD1, pP38 and VEGF (sc-40, sc-1265, sc-8404, sc-130348, sc-13073, sc-753, sc-7973, and sc-507, Santa Cruz Biotechnology). Secondary antibodies were: horseradish peroxidase-linked anti-rabbit and anti-mouse antibodies (Santa Cruz Biotechnology). The membranes were developed using Supersignal Ultra (Pierce).
Sc 507
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Sc-507 is a laboratory centrifuge designed for general-purpose applications. It can be used to separate and isolate various biological samples, such as cells, organelles, and macromolecules, based on their density differences. The centrifuge features adjustable speed and time controls to accommodate a range of sample types and processing requirements.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (2 mentions)
Sc 1506, by Santa Cruz Biotechnology (2 mentions)
Sc 40, by Santa Cruz Biotechnology (1 mentions)
P smad2, by Merck Group (1 mentions)
Sc 8404, by Santa Cruz Biotechnology (1 mentions)
Sc 13073, by Santa Cruz Biotechnology (1 mentions)
Sc 753, by Santa Cruz Biotechnology (1 mentions)
Sc 7973, by Santa Cruz Biotechnology (1 mentions)
Smad7, by R&D Systems (1 mentions)
Sc 1265, by Santa Cruz Biotechnology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Tgf β1, by Santa Cruz Biotechnology (1 mentions)
Sc 130348, by Santa Cruz Biotechnology (1 mentions)
P c jun, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Santa Cruz Biotechnology (1 mentions)
13 protocols using sc 507
1
Protein expression analysis by Western blot
2
Immunohistochemical Analysis of Ovarian VEGFA
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Ovaries were fixed at room temperature in Bouin’s solution, embedded in paraffin and sectioned at 5 μm according to standard procedures [6 (link)]. The rabbit polyclonal IgG VEGFA primary antibody was raised against a peptide corresponding to amino acids 1–140 of human VEGFA (catalog number: sc-507, Santa Cruz Biotechnology, Santa Cruz, CA). As a pan-antibody, it targeted both proangiogenic and antiangiogenic isoforms to confirm presence of VEGFA isoforms in KO and control mouse ovaries. The antibody was diluted 1:100 in 10% normal goat serum (NGS). As a negative control, serial sections were processed without primary antibody. Biotinylated goat anti-rabbit secondary antibodies were diluted 1:300 in 10% NGS and were used with each primary antibody in this study (catalog number: BA-1000, Vector Laboratories, Burlingame, CA). The secondary antibody was detected using aminoethyl carbazole (AEC) chromagen substrate solution (Invitrogen, Carlsbad, CA). Similar immunohistochemistry protocols were used for additional primary antibodies. The cytochrome P450, family 11, subfamily a, polypeptide 1 (CYP11A1) primary antibody (catalog number: ab78416; Abcam, Cambridge, Mass., USA) was diluted 1:200 in 10% normal goat serum in PBS) and was used to quantify the number of corpora lutea present.
3
Silencing HIF-1α in Mouse Brain Endothelial Cells
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bEnd3, an immortalized mouse brain endothelial cell line originally generated and characterized,46 (link) and lately used in our studies,47 (link) is commercially available at American Type Culture Collection ([ATCC] CRL-2299; Manassas, VA, USA). It is a well-characterized endothelial cell line that shows the expression of ICAM-1 and VCAM-1.47 (link) Cells were grown according to the supplier’s instructions and were allowed to grow to ~70%–80% confluence. HIF-1α expression was silenced for 72 hours by two different predesigned siRNA sequence primers from Santa Cruz Biotechnology Inc. (A: SC-44308 and B: SC-35562) and the corresponding scrambled siRNA (negative control) into the cells using the bEnd3 standard transfection reagent (CRL-2299; Alto-gen Biosystems, Las Vegas, ND, USA) according to the manufacturer’s instructions and as elsewhere described.48 (link) Transfected cells were treated with freshly prepared GSNO (100 μM) for 24 hours. Western blots for HIF-1α (ab65979; Abcam), VEGF (SC507; Santa Cruz Biotechnology Inc.), and β-actin (4967; Cell Signaling, Danvers, MA, USA) were performed using 20 μg protein samples.
4
VEGF Protein Expression Analysis in Lung Tissue
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The right upper lobes were homogenized in protein extraction solution. Protein quantification was performed by the BCA method (BCA Protein Assay kit). Samples containing 50 μg of protein were denatured, run on SDS-PAGE gel electrophoresis, and transferred to a nitrocellulose membrane. Protein expression was subsequently detected by incubation with rabbit polyclonal primary antibodies against VEGF (1:500; sc-507; Santa Cruz Biotechnology) at 4 C for 12 hours. Binding of the primary antibodies was detected with secondary antibodies conjugated to horseradish peroxidase, and the protein bands were detected by ImageJ software (National Institutes of Health) [14 (link)]. A work-flow scheme in the supporting information (S2 Fig ) indicates the analysis performed on each pulmonary lobe.
5
Comprehensive Cell Culture Protocol
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RPMI-1640 medium, 0.05% trypsin-EDTA, and penicillin-streptomycin solution were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Silibinin and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (Merck KGaA, St. Louis, MO, USA). Antibodies specific for VEGF (sc-507), STAT5b (sc-1656), CDK4 (sc-260), MMP9 (sc-13520), cyclin E (sc-481), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #2118) together with their specific secondary antibodies (anti-mouse (sc-516102) and anti-rabbit (sc-2357)) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies specific for p27 (#3686), p21 (#2974), phosphorylated epidermal growth factor (EGF) receptor (pEGFR; #3777), EGFR (#4267), phosphorylated JAK2 (pJAK2; (#3776) and JAK2 (#3230)), and phosphorylated STAT5 (pSTAT5; #9351) were purchased from Cell Signaling Technology (Beverly, MA, USA). Cyclin D1 (ab6152) antibody was obtained from Abcam (Cambridge, MA, USA). SOX2 (#MAB4423), NANOG (#MABD24), OCT4 (#MABD76), and MMP3 (#AB2963) were purchased from Merck Millipore (Burlington, MA, USA). The MMP2 (E90317) antibody was obtained from EnoGene (New York, NY, USA). The antibody specific for PD-L1 (R30949) was purchased from NSJ Bioreagents (San Diego, CA, USA).
6
Comprehensive Antibody and Inhibitor Protocol
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Antibodies against VEGF (SC-507), focal adhesion kinase (FAK; SC-932), Src (SC-5226), Akt (SC-5298), and CD34(SC-74499) were bought from Santa Cruz (Santa Cruz, CA, USA). Antibodies targeting p-FAK (3283S), p-Src (2101S), p-AKT (4060S), and CD133(64326s) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against CD31(ab28364) was purchased from Abcam (Cambridge, MA, USA). Small interfering RNAs (siRNAs) against FAK (L-003164-00), Src (L-003110-00) and Akt (L-003000-00-0005) and their respective controls were purchased from Dharmacon (Lafayette, CO, USA). We purchased VEGF shRNA plasmids from the National RNAi Core (Taipei, Taiwan). Inhibitors for FAK (869288-64-2) were from Calbiochem (San Diego, CA, USA). A VEGF ELISA kit (DY293B) was purchased from R&D Systems (Minneapolis, MN, USA) and APLN ELISA (KA1681) kit was purchased from Abnova (Taipei, Taiwan). Inhibitors for Src (P0042), Akt (A6730) and all the chemicals not mentioned above were supplied by Sigma-Aldrich (St. Louis, MO, USA).
7
Quantitative Western Blot Analysis
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Whole-cell extracts were prepared by resuspending cell pellets in a RIPA buffer supplemented with a cocktail of protease (Roche). The protein concentrations were quantified by the Bradford method. A total of 30 μg of protein extract was subjected to 10% SDS-PAGE gel electrophoresis, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), and blotted overnight with rabbit polyclonal anti-MMP-2 antibody (sc-10736, 1:800, Santa Cruz), rabbit polyclonal anti-VEGF antibody (sc-507, 1:350, Santa Cruz) and mouse monoclonal anti-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) antibody (sc-365062, 1:8000, Santa Cruz) in 5% BSA in Tris-buffered saline and 0.01% Tween-20. Peroxidase-conjugated secondary antibodies (sc-2371, 1:5000, Santa Cruz) were used and developed with the chemiluminescence reagent ECL Plus using hyperfilm (Amersham Biosciences). Quantification of the western blots was performed using Quantity One software. Each experiment was conducted three times, and a representative result was shown.
8
Protein Expression Analysis of Angiogenic Factors
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Tissue samples kept in liquid nitrogen were routinely processed for lysate preparation, protein loading, electrophoresis, transfer, and staining. Actin was used as the loading control. The protein loading volume was 30 μg in each well. The primary antibody concentration and exposure time of the blot were as follows: vascular endothelial growth factor (VEGF; 1 : 300, 3 min; sc-507, Santa Cruz Biotechnology, Inc., USA), fibroblast growth factor-basic (FGF2; 1 : 300, 3 min; sc-1360, Santa Cruz Biotechnology, Inc., USA), cluster of differentiation 31 (CD31; 1 : 200, 30 s; sc-1506, Santa Cruz Biotechnology, Inc., USA), and actin (1 : 5,000, 2 min; sc-1615, Santa Cruz Biotechnology, Inc., USA). All the secondary antibody concentrations were 1 : 2,000. As the molecular weights of VEGF (42 kDa) and actin (43 kDa) were similar, Restore™ Western Blot Stripping Buffer (21059; Thermo Fisher Scientific, Inc., USA) was used for actin blotting, and the washing time in the stripping buffer was 15 min. The blots were scanned and analyzed using software (iBright CL1500 Imaging System; Thermo Fisher Scientific, Inc., USA). The ratio of the target protein to actin in each sample in each group was compared 7 d and 14 d postsurgery.
9
Protein Expression Analysis in RPE/Choroidal Tissues
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RPE/choroidal tissues were isolated from eyecups and then lysed in Tissue Extraction Reagent I buffer (Invitrogen, Carlsbad, CA, USA) containing protease inhibitor cocktail according to the manufacturer's instructions. Total protein concentration was determined by bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL, USA). The same amount of protein (10 μg) was loaded in 4% to 15% precast polyacrylamide Tris-HCL gels (Bio-Rad, Hercules, CA, USA). After electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad) and then blocked with 5% skim milk for 1 hour. The membrane was incubated with primary antibody against VEGF (sc-507; Santa Cruz), VEGFR2 (#9698; Cell Signaling, Danvers, MA, USA), STC-1 (sc-30183; Santa Cruz), and β-actin (#4970; Cell Signaling) overnight at 4°C, and then washed and incubated with horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature. Proteins of interest were detected with enhanced chemilumescent reagents (Pierce Biotechnology) and the ratios of VEGF, VEGFR2, and STC-1 expressions to β-actin expression were determined. These values from the treatment groups were normalized to the average of the normal control group and expressed as a relative ratio for comparison.
10
Quantifying VEGFA Protein Expression
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