Following treatment in the presence or absence of MPs from all groups for 12 h at 37°C, aortic protein was harvested using RIPA lysis solution (Sigma-Aldrich; Merck KGaA). Protein concentrations were determined using a bicinchoninic acid protein assay. Subsequently, samples were treated with anti-eNOS antibody (cat. no. sc-136977; 1:1,000; Santa Cruz Biotechnology, Inc.) at 37°C for 24 h to immunoprecipitate eNOS. Then, the immunocomplex was mixed with Laemmli buffer (Sigma-Aldrich; Merck KGaA) and denatured (95°C for 5 min). After cooling on ice for ≥2 min, protein was obtained by centrifugation (680 × g, 2 min, 4°C). Then, immunoprecipitation of eNOS (cat. no. sc-654; 1:1,000; Santa Cruz Biotechnology, Inc.) and Hsp90 (cat. no. sc-13119; 1:1,000; Santa Cruz Biotechnology, Inc.) was tested as previously described (10 (link)). RPMI-1640 in the absence of MPs was set as a blank control.
Ripa lysis solution
Manufactured by Merck Group
Sourced in United States
RIPA lysis solution is a buffer used in the process of cell lysis, a technique used to break open cells and extract their contents, including proteins. The solution contains a combination of detergents, salts, and other agents that help solubilize and denature cellular components, allowing for the efficient extraction and isolation of proteins from cell samples.
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3 protocols using ripa lysis solution
1
Immunoprecipitation of eNOS and Hsp90
2
Immunoprecipitation Assay Protocol
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After removing the cells from the incubator and discarding the media, pre-cold PBS was used to wash the cells. After placing the cells on ice, add RIPA lysis solution (Sigma-Aldrich, USA), which contains a cocktail of protease and phosphatase inhibitors. For 30 min, cells were lysed on ice. Scrape off the cells with a cell scraper centrifuged at 16128 RCF/g for 20 min. Took 30 μL supernatant as Input group, add 5 × loading buffer, 100°C for 10 min. Added agarose beads to the supernatant (if agarose beads without antibodies, both antibodies and Protein G-Agarose should be used, and IgG antibodies and agarose beads are added to the control group), incubated for 6 h at 4°C on a vertical shaker for 10–12 rpm turnover. Finally, the samples were centrifuged at 4°C and 16128 RCF/g for 1 min, the supernatant was discarded, and agarose beads were washed with RIPA 5 times. Added 5×loading buffer, 100°C for 10 min, and the mixture was directly used for Western Blot detection.
3
Western Blot Protein Detection Protocol
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The cultured cells were harvested, and proteins were isolated using RIPA lysis solution (Sigma). Protein concentrations were determined using a BCA Protein Assay Reagent Kit (Beyotime). Equal amounts of protein were separated by precast 4–12% Bis-Tris gels (NuPage, Invitrogen) and transferred to NuPage 0.45 μm nitrocellulose membranes (Invitrogen). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h and incubated with primary antibodies overnight at 4 °C, and finally incubated with HRP-linked secondary antibodies for 1 h at room temperature. After incubation, the protein bands were visualized using an enhanced chemiluminescence detection system (SuperSignal West Pico, Thermo Scientific). All protein expression was normalized to the internal control, GAPDH.
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