B16F10 cells were first seeded in a 6-well plate at a density of 2 × 105 cells per well. After 4 h, the medium was discarded and the cells were treated with 2 mL of fresh medium containing PBS, AAV-PTENR, or AAV-PTEN [multiplicity of infection (MOI) = 1 × 105] for 48 h and then collected in a 2-mL tube and resuspended with 200 μL of lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% NP-40, 0.1% SDS, and protease inhibitor]. After the protein concentration was determined by the BCA Protein Assay Kit (Beyotime Biotechnology), a sample of 100 μg protein was run through an SDS-polyacrylamide gel electrophoresis gel and then transferred to a nitrocellulose membrane blot. Next, the membrane blots were soaked in blocking buffer [5% (w/v) nonfat milk, TBST buffer (50 mmol/L Tris-HCl, pH 7.6 and 150 mmol/L NaCl and 0.05% Tween 20)] for 1 h. Membrane blots were then incubated overnight in appropriate dilutions of primary antibody solution at 4 °C. Membrane blots were washed 3 times with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody at 1:5000 dilution for 1 h at 37 °C. Finally, protein signals were detected by an enhanced chemiluminescence detection system (Amersham/GE Healthcare) using a Typhoon Trio variable mode imager.
Enhanced chemiluminescence detection system
Manufactured by Cytiva
Sourced in United States, United Kingdom, Germany, Sweden, France, Japan
The Enhanced chemiluminescence detection system is a laboratory equipment that enables the detection and quantification of low-level luminescent signals in biological samples. It employs the principle of chemiluminescence, where a chemical reaction generates light that can be measured and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (22 mentions)
Polyvinylidene difluoride membrane, by Merck Group (18 mentions)
Nitrocellulose membrane, by Cytiva (13 mentions)
Protein assay kit, by Bio-Rad (11 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (11 mentions)
Gapdh, by Santa Cruz Biotechnology (8 mentions)
P akt, by Cell Signaling Technology (8 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (7 mentions)
β actin, by Santa Cruz Biotechnology (6 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Pvdf membrane, by Cytiva (6 mentions)
Anti β actin, by Merck Group (6 mentions)
Gapdh, by Abcam (6 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Chemidoc xrs imager system, by Bio-Rad (5 mentions)
Las 3000, by Fujifilm (5 mentions)
Anti akt, by Cell Signaling Technology (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Anti gapdh, by Abcam (5 mentions)
300 protocols using enhanced chemiluminescence detection system
1
Western Blot Analysis of PTEN Overexpression
2
Quantitative Western Blot Analysis
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Cell lysates were mixed with sample buffers and protease inhibitors in RIPA buffer, then heated at 100°C for 5 min. Abbreviations are expanded upon when first used. The language used is objective, concise, and technical terminology is consistent and accurate. After electrophoresis on an 8% SDS-polyacrylamide gel, the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were sealed before probing with the following antibodies: rabbit anti-SMAD4 (1:1000; Proteintech, China), rabbit anti-GLUT1 (1:1000; Proteintech, China), rabbit anti-HK2 (1:1000; Proteintech, China), and rabbit anti-GAPDH (1:1000; Proteintech, China). The bound antibodies were detected using a horseradish peroxidase (HRP)-coupled anti-rabbit secondary antibody (Proteintech, China) at a dilution of 1:3000 for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Bioscience, GE Healthcare, Piscataway, NJ, USA), and the membranes were exposed to X-ray film. The GAPDH antibody was used to confirm equal loading across gel lanes. The density of each protein band was then measured and quantified using ImageJ software.
3
Investigating AP-mediated Inflammation in BV2 Cells
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BV2 cells were seeded in a 6 cm dish at a density of 1 × 106 cells/dish and the AP extract (0.1 mg/mL) was added to the cells 1 h before incubation with or without LPS (1 μg/mL) for 0, 15, 30, or 60 min. Then, the supernatant was removed, and the cells were lysed with the radioimmunoprecipitation assay buffer on ice (cat. no. IBS-BR004; iNtRON Biotechnology, Sungnam, Republic of Korea). The lysates were boiled in 62.5 mM Tris-HCl buffer (pH 6.8) containing 2% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol. Then, proteins were separated into a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk in phosphate-buffered saline with Tween 20 for 2 h at room temperature, followed by incubation with primary antibodies (anti-phosphorylated p38 [1:1000; 9211], extracellular signal-regulated kinase (ERK)-1/2 [1:1000; 9101], and c-Jun N-terminal kinase (JNK) [1:1000; 9251], inhibitory κ Bα (Iκ Bα) [1:1000; 9212] antibodies) overnight at 4 °C. After washing thrice, the membrane was incubated with secondary antibodies horseradish peroxidase (HRP)-conjugated goat and anti-rabbit IgG [1:5000; sa002-500]) for 1 h at room temperature. Proteins were visualized using an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK), according to the manufacturer’s protocol.
4
Protein Expression Analysis by Western Blot
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Cells were harvested, lysed and sonicated in RIPA buffer. A total of 10-50 μg of protein was electrophoresed in SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were then incubated with the following antibodies: anti-ST8SIA4, anti-PI3K p110α, anti-phospho-Akt 308, anti-phospho-Akt 473, and anti-Akt and anti-NF-κB antibodies (Abgent, Cambridge, UK). The immunoblots were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences, Buckinghamshire, UK).
5
Colon Tissue Protein Analysis
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Protein samples were harvested from the colon tissues by Radio Immunoprecipitation Assay Lysis Buffer (P0013B, Beyotime) containing protease inhibitors. A bicinchoninic acid protein assay kit (ab102536, Abcam, Cambridge, MA, USA) was utilized to determine the protein concentration. Equal amounts of proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, P0670, Beyotime) prior to being transferred onto polyvinylidene fluoride (PVDF, FFP24) membranes, which were then blocked by 5% skim milk. Thereafter, these membranes were incubated with primary antibodies at 4°C for 24 h, including anti-tight junction protein 1 (ZO-1, 1/1000, ab216880, Abcam), anti-Claudin-1 (1/2000, ab180158, Abcam) and anti-β-actin (1 µg/ml, ab8226, Abcam) antibodies. Subsequently, these membranes underwent 1-h cultivation with HRP-conjugated goat anti-rabbit secondary antibody (1/2000, ab6721, Abcam) or goat anti-mouse secondary antibody (1/2000, ab205719, Abcam) for visualization. β-actin was regarded as an internal reference. The protein band was obtained by an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA).
6
Immunoblot Analysis of Epidermal Differentiation Markers
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Following treatment with stimulatorα, HaCaT cells were harvested and lysed in lysis buffer. Samples were separated by performing 10% SDS-PAGE and then transferred to nitrocellulose membrane. Blots were incubated with antibodies against filaggrin, phospho-JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), involucrin, or loricrin (Proteintech, Rosemont, IL, USA). After incubation, the membrane was developed by using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
7
Western Blot Analysis of Proteins
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Total proteins were extracted using RIPA buffer containing protease inhibitor cocktail (Roche, Basel, Switzerland). The concentrations of extracted proteins were determined using BCA Protein Quantification Kit (Beyotime, Shanghai, China). A total of 20 μg protein from each treatment was separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto the polyvinylidene fluoride (PVDF) membranes. Then the membranes were incubated with primary antibodies against RAP1A, MMP9, vimentin, and GAPDH overnight at 4°C. anti-RAP1A (ab115776), anti-MMP9 (ab58803), anti-vimentin (ab137321), and anti-GAPDH (ab181603) were purchased from Abcam (Cambridge, MA, USA) and used at the following dilutions: anti-RAP1A (1:500), anti-MMP9 (1:500), anti-vimentin (1:500), and anti-GAPDH (1:1,000). After being incubated with horseradish peroxidase-labeled secondary antibody at 37°C for 1 hour, the blots were developed using the enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK),and the blots were quantified using Image J software (NIH, Bethesda, MD, USA).
8
Western Blot Analysis of Apoptosis Markers
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Cell lysate was boiled in a sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol), and protein concentration was determined using a Bradford protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin as the standard [47 (link)]. Following protein transfer, the membrane was blocked with 5% skim milk in PBS Tween- (PBST-) 20 for 2 h at room temperature and then incubated overnight with antibodies at 4°C (the primary antibodies include Bcl2, 1 : 1000, Cell Signaling Technology, #3498; Bax, 1 : 1000, Cell Signaling Technology, #2772; caspase-9, 1 : 1000, Cell Signaling Technology, #9504; c-IAP, 1 : 1000, Cell Signaling Technology, #4952). The membranes were then washed with PBST containing 0.1% Tween. After three washes in PBST, each blot was incubated with peroxidase-conjugated secondary antibody for 1 h at 37°C. Labeled proteins were visualized using the Odyssey infrared scanner (LI-COR, Lincoln, NB, USA) [48 (link)]. Signals were densitometrically assessed and normalized to the β-actin signals, and an enhanced chemiluminescence detection system (Amersham, Piscataway, NJ, USA) was used to visualize the antibody-specific proteins in accordance with the manufacturer's recommended protocol [49 (link)].
9
Immunoprecipitation and Western Blotting
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For immunoprecipitation, cellular lysates were incubated with antibody overnight, followed by incubating with protein A/G-sepharose beads (Santa Cruz Biotechnology) for 4 h; 2500 × g centrifugation and three times washing with ice-cold lysis buffer. The immunoprecipitated proteins were eluted by denaturation Laemmli buffer at 95 ℃ for 5 min; separated by SDS-PAGE gel, transferred to a PVDF membrane (Pierce), blocked with 5% nonfat milk and incubating with primary antibodies, then secondary antibodies. The protein was finally visualized using enhanced chemiluminescence detection system (Amersham Biosciences Europe, Freiberg, Germany) according to the manufacturer’s instructions.
10
Arterial Protein Isolation and Analysis
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For the isolation of proteins from arterial tissues, the samples were lysed by the addition of SDS-PAGE sample buffer followed by homogenization. After transfer to PVDF membranes (Millipore, Darmstadt, Germany), proteins were incubated with primary antibodies using anti-collagen I (Abcam, ab21286, Cambridge, UK), NOTCH1, 2, and 3 (Cell Signaling, Danvers, MA, USA). Secondary antibodies specific for peroxidase-conjugated anti-mouse IgG (Sigma-Aldrich; 1:10,000, St. Louis, MO) or anti-rabbit IgG (Sigma-Aldrich; 1:5000, St. Louis, MO) were used as needed. Blots were visualized using an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK). Samples were normalized to GAPDH (Santa Cruz; 1:10,000, SC-32233, CA, USA) and quantified by densitometry.
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