Heart tissues (20 mg) were thoroughly homogenized with a plastic grinding rod at 4 °C. Nuclear protein was extracted according to the instructions and quantified by a BCA assay. The gels were 12% separation and 5% spacer gels. The same amount of protein from different samples was mixed well with 5× SDS protein loading buffer at a ratio of 4:1 and denatured at 100 °C for 5–10 min. Then, the proteins were loaded onto SDS-polyacrylamide gels for electrophoresis. The proteins were run on the spacer gel at 80 V and on the separation gel at 100 V and blotted onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked in 5% bovine serum albumin for 60 min at room temperature. The blocked PVDF membranes were placed in diluted primary antibody (H3K9, Abcam, UK; Lamin B1, Boster, China) overnight at 4 °C, washed, placed in diluted secondary antibody (HRP-linked antibody, Merck, Germany), and incubated for 1 h at room temperature. The PVDF membranes were placed in the reaction solution of a chemiluminescence kit, incubated for 2 min at room temperature and exposed to film. The film was scanned and saved, and grayscale analysis was performed using Quantity One software to calculate the gray value of Mef2C/Lamin B1 for each group.
Lamin b1
Manufactured by Boster Bio
Sourced in United Kingdom, United States
Lamin B1 is a protein that is a structural component of the cell nucleus. It is a member of the lamin protein family and plays a role in the organization and maintenance of the nuclear lamina, which provides structural support and organization to the cell nucleus.
Automatically generated - may contain errors
2 protocols using lamin b1
1
Western Blot Analysis of H3K9 and Lamin B1
2
Oleocanthal Modulates Inflammatory Signaling in Chondrocytes
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Primary human OA chondrocytes were plated in 12-well plates, stimulated with oleocanthal 1-5 µM 12 hours and then incubated with LPS (250 ng/mL) during 24 hours or 30 minutes.
Proteins were extracted using a lysis buffer with a commercial protease inhibitor cocktail (Thermo Fisher). SDS-PAGE and blotting procedure were carried on as previously described [13] . Immunoblots were incubated with the appropriate antibody (iNOS, IkB, p-ERK-1/2 and NF-kB p65 from Cell Signaling MA, USA; COX-2 from DAKO, Denmark; MMP13 from Santa Cruz Biotechnology, USA; ERK1/2 and p-38 from Upstate, p-p38 from Millipore; Lamin B1 from Boster, CA, USA) and visualized with an Immobilon Western Detection kit (Millipore Massachusetts, USA) using horseradish peroxidase-labelled secondary antibody. To confirm equal loading in each sample, the membranes were stripped in glycine buffer at pH 2 and re-blotted with an anti-GAPDH antibody (Sigma). The images were captured and analysed with an EC3 imaging system (UVP). Data obtained were further validated by densitometric analysis using Image J software.
Proteins were extracted using a lysis buffer with a commercial protease inhibitor cocktail (Thermo Fisher). SDS-PAGE and blotting procedure were carried on as previously described [13] . Immunoblots were incubated with the appropriate antibody (iNOS, IkB, p-ERK-1/2 and NF-kB p65 from Cell Signaling MA, USA; COX-2 from DAKO, Denmark; MMP13 from Santa Cruz Biotechnology, USA; ERK1/2 and p-38 from Upstate, p-p38 from Millipore; Lamin B1 from Boster, CA, USA) and visualized with an Immobilon Western Detection kit (Millipore Massachusetts, USA) using horseradish peroxidase-labelled secondary antibody. To confirm equal loading in each sample, the membranes were stripped in glycine buffer at pH 2 and re-blotted with an anti-GAPDH antibody (Sigma). The images were captured and analysed with an EC3 imaging system (UVP). Data obtained were further validated by densitometric analysis using Image J software.
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