Proteins from cell lysates at equal loadings were subjected to 7.5% to 12.5% SDS‐PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were further blocked in 5% nonfat milk, followed by incubation with the corresponding primary antibodies overnight at 4°C and with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature. Blots were detected using an electrochemiluminescence system (GE Healthcare Biosciences) and qualified with Quantity One (BioRad) software. We also detected β‐actin as the protein loading control.
Electrochemiluminescence system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Electrochemiluminescence system is a laboratory instrument used to detect and measure the presence and concentration of specific molecules or analytes in a sample. The system utilizes electrochemical reactions to generate light, which is then detected and quantified to provide analytical data. The core function of the Electrochemiluminescence system is to perform sensitive and accurate assays for a variety of applications, including drug development, biomarker detection, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Proteome profiler angiogenesis array, by R&D Systems (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Pdcd4 antibody, by Cell Signaling Technology (1 mentions)
Tween 20, by Nacalai Tesque (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
β actin antibody, by MP Biomedicals (1 mentions)
Las 4000 mini imaging system, by Fujifilm (1 mentions)
Anti twist, by Abcam (1 mentions)
Anti src, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti hoxb7, by Abcam (1 mentions)
Horseradish peroxidase conjugated, by Beyotime (1 mentions)
Total protein extraction kit, by Keygene (1 mentions)
Bicinchoninic acid protein assay, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Vegf a antibody, by Abcam (1 mentions)
8 protocols using electrochemiluminescence system
1
Western Blot Analysis of Protein Expression
2
Angiogenesis Protein Profiling of Scaffold Medium
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After a 72-h incubation of the rolled scaffold in exogenous growth factor-free medium, the medium was collected and used in the Proteome Profiler Angiogenesis Array (R&D Systems, MN, USA), according to manufacturer’s instructions. Briefly, nitrocellulose membranes were blocked in blocking buffer (1 h at room temperature). Samples were diluted in array buffer and mixed with detection antibody cocktail (biotin labeled against different proteins), followed by an overnight incubation on the membrane (4°C). Following overnight incubation, membranes were washed three-times in wash buffer and subsequently incubated in horseradish peroxidase (HRP)-conjugated streptavidin complex for 30 min (room temperature). After washing, signal was detected by electrochemiluminescence system (GE Healthcare, IL, USA). The signal density of each blot was then determined by densitometry using ImageJ software (NIH, MD, USA).
3
Western Blot Analysis of Nuclear and Cytoplasmic Proteins
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Protein samples extracted from heart tissues or cultured cells were subjected to Western blot analysis, as described previously.17 The nuclear and cytoplasmic extractions of the cell samples were performed using the NE‐PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL) following the manufacturer's instructions. Antibodies against GAPDH or proliferating cell nuclear antigen (PCNA) and tubulin served as controls for normalization of nuclear and cytosolic fractions, respectively. Specific antibody binding was visualized and quantified by an electrochemiluminescence system (GE Healthcare).
4
Western Blot Analysis of miR-21 Regulation
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After transfection with miR-21 mimic or inhibitor for 48 h, cells were harvested and lysed using RIPA buffer (Cell Signaling Technology, Dancers, MA, USA) supplemented with phenylmethylsulphonyl fluoride (Nacalai Tesque, Kyoto, Japan). Equal amounts of protein were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (0.45 μm; Millipore). Membranes were washed with Tris-buffered saline (TBST) containing 0.1% Tween-20 (Nacalai Tesque) and 5% bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA), and incubated overnight at 4°C with primary antibodies (PDCD4 antibody from Cell Signaling Technology, Danvers, MA, USA; β-actin antibody from MP Biomedicals, LLC, Irvine, CA, USA). After washing with TBST, membranes were further incubated with horseradish peroxidase-conjugated secondary antibody (1:10 000; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature and developed by using an electrochemiluminescence system in the end (GE Healthcare, Little Chalfont, UK). Protein bands were detected by an LAS4000mini imaging system (Fujifilm, Tokyo, Japan), and band intensities of western blots were quantitatively measured by calculating integrated grayscale densities in consistently sized windows incorporating each band using ImageJ version 1.48 software, as described previously [22 (link)] .
5
Western Blot Analysis of Signaling Proteins
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Total protein levels in lysis supernatant of SGC7901 and SNU1 were determined with a BCA protein assay kit (Thermo Fisher Scientific). Total protein (25 μg) was separated on 10% and 15% SDS-PAGE. Electrophoretically pure was transferred onto nitrocellulose membranes (Millipore),and incubated with primary antibodies at 4°C overnight, followed by a secondary antibody for another hour at 25°C. Immunoreactive bands were analyzed with an electrochemiluminescence system (GE Healthcare).
Primary antibodies used in our study were: anti-HOXB7 (Abcam ab168466), anti-Src (Cell Signaling Technology [CST] 2108), anti-p-Src-Y416 (CST 2101), anti-FAK (CST 3285), antip-FAK-Y397 (CST 8556), anti-FAK-phospho-Y576+Y577 (CST 3281), GAPDH (CST 5174), anti-E-cadherin (CST 14472), anti-N-cadherin (CST 4061), antivimentin (CST 5741), anti-Twist (Abcam Ab49254), anti-β-catenin (CST 8480). Secondary antibodies used in our study were horseradish peroxidase–conjugated (Beyotime).
Primary antibodies used in our study were: anti-HOXB7 (Abcam ab168466), anti-Src (Cell Signaling Technology [CST] 2108), anti-p-Src-Y416 (CST 2101), anti-FAK (CST 3285), antip-FAK-Y397 (CST 8556), anti-FAK-phospho-Y576+Y577 (CST 3281), GAPDH (CST 5174), anti-E-cadherin (CST 14472), anti-N-cadherin (CST 4061), antivimentin (CST 5741), anti-Twist (Abcam Ab49254), anti-β-catenin (CST 8480). Secondary antibodies used in our study were horseradish peroxidase–conjugated (Beyotime).
6
Protein Expression Analysis in Cells
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Cells were digested with the Total Protein Extraction Kit (KeyGene, China) and centrifuged to collect cell supernatants. A BCA assay was used to determine the protein concentration. Proteins (30 μg) were separated on 10% sodium dodecyl sulphate-polyacrylamide gel and transferred to a PVDF membrane, and then blocked with 5% nonfat dry milk dissolved in TBST (Tris-buffered saline, 0.1% Tween-20). The primary antibodies anti-DMP1, anti-DSPP, anti-OPN, anti-ALP, anti-RUNX2, and GAPDH were used at dilutions of 1:1000 for 2 h at room temperature, and then washed with TBST twice on a TS rocker. Secondary antibodies were applied in the same way. Bands were monitored with an electrochemiluminescence system (GE, USA).
7
Western Blot Protein Analysis Protocol
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The samples were lysed on ice in Western and IP Cell Lysis Buffer, and the protein concentration was determined using the bicinchoninic acid protein assay (Beyotime). SDS-PAGE was used to separate protein samples. The membrane was blocked with 5% skim milk before overnight incubation with primary antibodies at 4 °C. All primary antibodies were diluted at a ratio of 1:1000 except anti-alpha tubulin which was diluted at 1:5000. Horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution, Jackson ImmunoResearch) and an electrochemiluminescence system (GE Healthcare, UK) were used to detect specific proteins. Western blot results were quantified with Fiji (version 1.53c). All antibody information is listed in Supplementary Table 3 .
8
Western Blot Analysis of Signaling Proteins
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Proteins from cell lysates at equal loadings were subjected to 7.5% to 12.5% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were further blocked in 5% nonfat milk, followed by incubation with the corresponding primary antibodies overnight at 4°C and with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The PI3K antibody (abcam,1:1000), Akt antibody (abcam,1:1000), VEGFR2 antibody (abcam,1:1000), VEGFA antibody (abcam,1:1000), Blots were detected using an electrochemiluminescence system (GE Healthcare Biosciences) and qualified with Quantity One (Bio-Rad) software. We also detected β-actin as the protein loading control.
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