Western blot analysis was performed as previously described 31 (link). Briefly, MDSCs were lysed in Cell Lytic MT lysis buffer (Sigma) with Protease Inhibitor Cocktail (Invitrogen) and phosphatase inhibitor 2 and 3 (Sigma) for 15 min on a shaker. After centrifugation for 20 min at 12 000×g (4°C), the supernatants were saved and protein concentrations of the samples were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 μg) were loaded onto SDS-polyacrylamide gels and blotted onto PVDF membranes (BioRad). Western blot analysis was performed using antibodies against mTOR, phospho-mTOR, p70S6K, phospho-p70S6K, S6, and phospho-S6 (rabbit monoclonal antibodies, 1: 1 000, Cell Signaling). Antibody against β-actin (rabbit monoclonal anti-β-actin, 1: 2 000, Cell Signaling) was used as a loading control. For detection, the membrane was incubated with anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (1: 2 000, Cell Signaling). Bands were visualized using SuperSignal West Pico Chemiluminescent substrate (ThermoScientific Pierce).
Phosphatase inhibitor 2 and 3
Manufactured by Merck Group
Sourced in United States
Phosphatase inhibitor 2 and 3 are lab equipment products used to inhibit the activity of phosphatases, which are enzymes that remove phosphate groups from various molecules. These inhibitors are commonly used in biochemical research, cell biology, and protein analysis applications to maintain the phosphorylation state of proteins, which is important for understanding cellular signaling pathways and regulatory mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Rabbit monoclonal anti β actin, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Cell lytic mt lysis buffer, by Merck Group (2 mentions)
Anti rabbit igg secondary antibodies conjugated with horseradish peroxidase, by Cell Signaling Technology (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
5 protocols using phosphatase inhibitor 2 and 3
1
Western Blot Analysis of mTOR Pathway
2
Protein Extraction and Western Blot Analysis
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Total protein from mouse cortex was extracted using RIPA buffer including phosphatase inhibitor 2 and 3 (Sigma‐Aldrich, St. Louis, MO, USA) and a protease inhibitor cocktail tablet (Roche Diagnostic, Rotkreuz, Switzerland) and total protein from cell experiments was extracted using M‐PER including 2% SDS and DTT. Protein was then separated on a 12% Criterion TGX Stain‐free gel and proteins were transferred to a nitrocellulose membrane, which was blocked with 5% skimmed milk in PBS‐Tween. The membrane was washed in PBS‐Tween and incubated with specific primary antibodies (see Table 3 for target and dilution). Next, the membrane was washed with PBS‐Tween and incubated with the appropriate secondary antibody. Finally, the membrane was incubated with the detection reagent ECL‐Prime (GE‐healthcare, Chicago, IL, USA) and processed in the Western Blot Imager (ChemiDoc MP, Bio‐Rad, CA, USA). Detected protein was normalized against total protein levels.47
3
Extracellular Vesicle Lysis and Protein Extraction
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Isolated sEVs were resuspended in the lysis buffer with a volume ratio of 4:1 (fraction/buffer) with a final concentration of 20 mM HEPES, pH 8.0, 5% glycerol, 0.1% n-Dodecyl-β-D-Maltoside, 0.2 mM DTT, 1.6 M urea, and 1/250 (v:v) phosphatase inhibitors (phosphatase inhibitor 2 and 3; Sigma: Cat No. P0044 and P5726) and gently vortexed for 3 min. The suspension was centrifuged for 1 min at 10,000× g, and the supernatant was then collected.
4
Western Blot Analysis of mTOR Pathway
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Western blot analysis was performed as previously described 31 (link). Briefly, MDSCs were lysed in Cell Lytic MT lysis buffer (Sigma) with Protease Inhibitor Cocktail (Invitrogen) and phosphatase inhibitor 2 and 3 (Sigma) for 15 min on a shaker. After centrifugation for 20 min at 12 000×g (4°C), the supernatants were saved and protein concentrations of the samples were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 μg) were loaded onto SDS-polyacrylamide gels and blotted onto PVDF membranes (BioRad). Western blot analysis was performed using antibodies against mTOR, phospho-mTOR, p70S6K, phospho-p70S6K, S6, and phospho-S6 (rabbit monoclonal antibodies, 1: 1 000, Cell Signaling). Antibody against β-actin (rabbit monoclonal anti-β-actin, 1: 2 000, Cell Signaling) was used as a loading control. For detection, the membrane was incubated with anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (1: 2 000, Cell Signaling). Bands were visualized using SuperSignal West Pico Chemiluminescent substrate (ThermoScientific Pierce).
5
Western Blot Protein Expression Analysis
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Protein expression was determined using Western blotting technique as previously described [34 (link)]. Briefly, cells were seeded in 6-well plates and incubated until 70–80% confluence. At the end of the treatment, cells were washed once with ice-cold 1X PBS and whole-cell lysates were prepared by homogenizing in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor 2 and 3 (Sigma-Aldrich). Protein concentration was determined by Bradford method and equal amount of protein was resolved by SDS polyacrylamide gel electrophoresis. The proteins were then transferred on to nitrocellulose membrane and probed with specific primary antibody. Blots were then incubated with species-specific fluorescent-tagged IgG secondary antibody and scanned and visualized using LI-COR Odyssey® imaging system (LI-COR Biosciences). Each band was normalized to the corresponding total STAT3 or β-actin bands. Densitometric analysis of the bands was performed using ImageJ software and results were plotted as percent of control where control was considered as 100%.
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