Protein was isolated from rBMSCs treated with leptin with 103 ng/ml using a Beyotime Cell Protein Extraction kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The total concentration of protein was determined using the bicinchoninic acid assay method. Proteins (20 µg/ml; 0.6 µg/lane) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked for 30 min with 5% fat-free milk at room temperature and incubated with the following primary antibodies: Anti-ERK1/2 (1:1,000), anti-p-ERK1/2 (1:800), anti-JNK (1:1,500), anti-p-JNK (1:1,000), anti-p38 (1:500), anti-p-p38 (1:800), anti-p90rsk (1:1,000) and anti-p-p90rsk (1:1,000) for a minimum of 1 h at 37°C. Membranes were subsequently incubated with horseradish-conjugated (H+L) secondary antibodies (1:4,000) at 37°C and developed using a Pierce™ enhanced chemiluminescence plus western blotting substrate (cat. no. 32132; Thermo Fisher Scientific, Inc.). UVP VisionWorksLS software (UVP, LLC; DBA Analytik Jena US, Upland, CA, USA).
Uvp visionworksls software
Manufactured by Analytik Jena
Sourced in United Kingdom
UVP VisionWorksLS software is a comprehensive imaging and analysis platform designed for use with Analytik Jena's UV transilluminators and imaging systems. The software provides tools for capturing, processing, and analyzing images of gels, blots, and other samples illuminated by UV light.
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Lab products found in correlation
Cell protein extraction kit, by Beyotime (1 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Lysis solution, by Thermo Fisher Scientific (1 mentions)
Bradford assay kit, by Takara Bio (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti phosphorylated extracellular signal regulated kinase p erk 1 2, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti phosphorylated p53, by Cell Signaling Technology (1 mentions)
Anti gpx4, by Abcam (1 mentions)
4 protocols using uvp visionworksls software
1
Protein Analysis of Leptin-Treated rBMSCs
2
Protein Extraction and Western Blot Analysis
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Certain collected tissues were homogenized in RIPA buffer (50 mM Tris HCl pH 7.4, 50 mM NaCl, 5 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1% aprotinin, 50 mM NaF, 0.1 mM Na3VO4) then centrifuged to collect cell lysate supernatants. The procedures of SDS-PAGE and protein transfer, and the antibodies used, were as described in our earlier report [48 (link)]. Digital images were obtained using a luminescence reader (Biospectrum-UVP, Cambridge, UK) and densitometry analysis was performed using UVP VisionWorks LS software (UVP, Cambridge, UK).
3
Western Blot Analysis of Akt and PI3K
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Cells were lysed in lysis solution (Ambion, Grand Island, NY, USA) and incubated at 95 °C for 10 min. Protein concentration was determined by the Bradford assay kit (Takara Biotechnology, Dalian, China). Twenty micrograms of total proteins was separated by 10%–12% sodium dodecyl sulfate polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. Blots were probed with rabbit monoclonal anti-Akt (1:1000), anti-pAkt (1:1000), anti-PI3K (1:1000), anti-pPI3K (1:1000). Blots were also probed with rabbit monoclonal anti-GAPDH antibody (Milwaukee, WI, USA, Sigma, 1:10,000) as a loading control. Anti-rabbit secondary antibodies conjugated to horseradish peroxidase were used at 1:10,000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). UVP BioSpectrum®CCD imaging system (Davis, CA, USA) was used for imaging and analysis. Camera settings were manipulated in preview mode to optimize the exposure and determine the appropriate final exposure settings. Exposures of 30 s up to 5 min were used for data collection. Results were analyzed through scanning densitometry by UVP Vision Works LS Software (UVP, Cambridge, UK).
4
Western Blot Analysis of Apoptosis Signaling
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Samples from cells and animals were lysed in RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor cocktail. The protein concentration was detected by BCA kits (Thermo Fisher Scientific, USA). Samples were separated by SDS-poly acrylamide gel electrophoresis and transferred to PVDF membrane. After blocked with 100 g/L non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibody overnight at 4 °C, and then with secondary antibodies coupled to horseradish peroxidase. The primary antibodies were used for Western blot analysis:
anti-phosphorylated extracellular signal-regulated kinase (p-Erk) 1/2, anti-Erk1/2, antiphosphorylated p53, anti-p53 and anti-β-actin were purchased from Cell Signaling Technology (Boston, MA). Anti-Gpx4, anti-Bax, anti-Bcl-2 and anti-cleved caspase-3
were purchased from Abcam (UK). The secondary antibodies were used for Western blot analysis: anti-rabbit IgG-HRP and anti-mouse IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA). Cross-reactivity was visualized using ECL Western blotting detection reagents and analyzed by scanning densitometry using a UVP Vision Works™ LS Software (UVP, Cambridge, UK) and quantified with ImageJ Software.
anti-phosphorylated extracellular signal-regulated kinase (p-Erk) 1/2, anti-Erk1/2, antiphosphorylated p53, anti-p53 and anti-β-actin were purchased from Cell Signaling Technology (Boston, MA). Anti-Gpx4, anti-Bax, anti-Bcl-2 and anti-cleved caspase-3
were purchased from Abcam (UK). The secondary antibodies were used for Western blot analysis: anti-rabbit IgG-HRP and anti-mouse IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA). Cross-reactivity was visualized using ECL Western blotting detection reagents and analyzed by scanning densitometry using a UVP Vision Works™ LS Software (UVP, Cambridge, UK) and quantified with ImageJ Software.
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