BMDMs were pretreated with porous Se@SiO2 nanospheres (Se@SiO2 group; 10 µg/mL) or M-Se@SiO2 (M-Se@SiO2 group; 10 µg/mL) for 1 h, and then LPS was added (100 ng/mL). After 30 min, the cells were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors (EpiZyme, China). The extracted total proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes. The membranes were blocked in a blocking solution (Beyotime) for 15 min, washed, and incubated with primary antibodies overnight at 4 °C. Antibodies specific for p38/p-p38, ERK/p-ERK, and p65/p-p65 (1:1000, Cell Signaling, USA) were used in the experiments. After washing, the membranes were incubated with a goat anti-rabbit IgG horseradish peroxidase-conjugated antibody (1:1000, Cell Signaling, USA) for 1 h at room temperature. The blots were developed with an enhanced chemiluminescent reagent (Millipore, USA) and the Tanon Imaging System (Tanon, China) for chemiluminescent detection. Relative band intensities were quantified using ImageJ software, and all results were normalized to β-actin expression.
Protease and phosphatase inhibitors
Manufactured by Ipsen
Sourced in China
Protease and phosphatase inhibitors are laboratory reagents used to prevent the breakdown of proteins and phosphorylated molecules during sample preparation and analysis. They help maintain the integrity of target proteins and signaling pathways within biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Blocking solution, by Beyotime (2 mentions)
Enhanced chemiluminescent reagent, by Merck Group (2 mentions)
Ripa lysis buffer, by Ipsen (2 mentions)
Ab134045, by Abcam (2 mentions)
Ab92726, by Abcam (2 mentions)
Anti par1 26366 1 ap, by Proteintech (2 mentions)
Anti slug 12129 1 ap, by Proteintech (2 mentions)
Anti snai1 13099 1 ap, by Proteintech (2 mentions)
Tanon 5200multi device, by Merck Group (2 mentions)
Amersham hybond 0.45 m, by GE Healthcare (2 mentions)
Anti zeb 1 21544 1 ap, by Proteintech (2 mentions)
Anti gapdh 60004 1 ig, by Proteintech (2 mentions)
Anti e cadherin 20874 1 ap, by Proteintech (2 mentions)
Anti vimentin 10366 1 ap, by Proteintech (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Goat anti rabbit igg horseradish peroxidase, by Cell Signaling Technology (1 mentions)
4 protocols using protease and phosphatase inhibitors
1
Evaluating Selenium-Enriched Nanospheres' Impact on Macrophage Signaling
2
Exosome protein profiling by Western blot
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Cell and exosome samples were lysed using RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Epizyme, Shanghai, China). The protein concentration was quanti ed using a BCA protein assay kit (Beyotime). Proteins were separated by 10% (gradient) SDS-PAGE (Epizyme) and transferred onto PVDF membranes (Amersham Hybond 0.45µm; GE Healthcare). The membranes were blocked with 5% milk in trisbuffered saline-Tween (TBS-T) for 2h, then incubated with primary antibodies (anti-CD9(ab92726,Abcam),anti-CD63(ab134045,Abcam), anti-MMP-1(ab134184,Abcam), anti-PAR1(26366-1-AP,Proteintech),anti-Zeb-1(21544-1-AP,Proteintech), anti-Slug(12129-1-AP,Proteintech), anti-SNAI1(13099-1-AP,Proteintech), anti-E-cadherin(20874-1-AP,Proteintech), anti-Vimentin(10366-1-AP,Proteintech), anti-GAPDH(60004-1-Ig,Proteintech)) for 12h at 4°C. the membranes were washed with TBS-T for 3times,15min per time, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT. The proteins were visualized using the ECL western blotting substrate (cat. WBKLS0100, Millipore) and a Tanon-5200Multi device.
3
Western Blotting Analysis of Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were pretreated with Se@SiO 2 or M-Se@SiO 2 for 1 h, and then LPS was added (100 ng/mL). After 30 min, the cells were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors (EpiZyme, China). The extracted total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes. The membranes were blocked in blocking solution (Beyotime) for 15 min, washed, and incubated with primary antibodies overnight at 4 °C. p38/p-p38, extracellular signal-regulated kinase (ERK)/p-ERK, and p65/p-p65 antibodies (1:1000, Cell Signaling, USA) were used for the experiments. After washing, the membranes were incubated with goat antirabbit IgG-horseradish peroxidase (1:1000, Cell Signaling, USA) for 1 h at room temperature. Blots were developed with enhanced chemiluminescent reagent (Millipore, USA) and Tanon Imaging System (Tanon, China) for chemiluminescent detection. Relative band intensities were quantified using ImageJ software, and all results were normalized to β-actin expression.
4
Exosome protein profiling by Western blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell and exosome samples were lysed using RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Epizyme, Shanghai, China). The protein concentration was quanti ed using a BCA protein assay kit (Beyotime). Proteins were separated by 10% (gradient) SDS-PAGE (Epizyme) and transferred onto PVDF membranes (Amersham Hybond 0.45µm; GE Healthcare). The membranes were blocked with 5% milk in trisbuffered saline-Tween (TBS-T) for 2h, then incubated with primary antibodies (anti-CD9(ab92726,Abcam),anti-CD63(ab134045,Abcam), anti-MMP-1(ab134184,Abcam), anti-PAR1(26366-1-AP,Proteintech),anti-Zeb-1(21544-1-AP,Proteintech), anti-Slug(12129-1-AP,Proteintech), anti-SNAI1(13099-1-AP,Proteintech), anti-E-cadherin(20874-1-AP,Proteintech), anti-Vimentin(10366-1-AP,Proteintech), anti-GAPDH(60004-1-Ig,Proteintech)) for 12h at 4°C. the membranes were washed with TBS-T for 3times,15min per time, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT. The proteins were visualized using the ECL western blotting substrate (cat. WBKLS0100, Millipore) and a Tanon-5200Multi device.
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