After incubation with PBS, OA-RD17 (1 nM), lipopolysaccharide (LPS, 1 μg/mL) (Solarbio, China), specific TLR4 inhibitor (1 μg/mL), miR-632 mimic (50 nM), or miR-632 inhibitor (100 nM) for 24 h, respectively, cell lysates (RIPA: PMSF: phosphatase inhibitor = 100:1:1; RIPA and PMSF, Meilun Biotechnology, Dalian, China; phosphatase inhibitor, Roche, Shanghai, China) were used to extract total protein in keratinocytes and macrophages. Moreover, cell lysates were also used to extract total protein in wound tissues from SD rats. The extracted proteins were quantified using the Bradford method (BCA protein analysis kit, Meilun, Dalian, China). The protein samples were then separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electro-imprinted on polyvinylidene fluoride membranes, and recorded and analyzed quantitatively using the Bole exposure software system. Primary antibodies, including GAPDH, Lamin B1, P38, P-P38, ERK, P-ERK, JNK, P-JNK, IκB, P-IκB, P65, P-P65 (Affinity, China), GSK3β, β-catenin, Cyclin D1, c-MYC, and Vimentin (ZEN BIO, China) were used following the provided instructions.
Radioimmunoprecipitation assay (ripa)
Manufactured by Meilun
Sourced in China
RIPA is a laboratory reagent used for the extraction and solubilization of proteins from biological samples. It facilitates the disruption of cell membranes and the release of intracellular proteins, making them available for subsequent analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg hrp, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Lipopolysaccharide lps, by Solarbio (1 mentions)
P erk, by Affinity Biosciences (1 mentions)
P jnk, by Affinity Biosciences (1 mentions)
Bca protein analysis kit, by Meilun (1 mentions)
C myc, by ZenBio (1 mentions)
Cyclin d1, by ZenBio (1 mentions)
Lamin b1, by Affinity Biosciences (1 mentions)
β catenin, by ZenBio (1 mentions)
Vimentin, by ZenBio (1 mentions)
P iκb, by Affinity Biosciences (1 mentions)
Gapdh, by Affinity Biosciences (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Meilun (1 mentions)
Raloxifene, by Meilun (1 mentions)
Malondialdehyde, by Beyotime (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
Cell counting kit 8 cck 8, by Keygen Biotech (1 mentions)
Hrp labeled secondary antibody, by Proteintech (1 mentions)
3 protocols using radioimmunoprecipitation assay (ripa)
1
Protein Expression Analysis in Wound Healing
2
Quantitative Protein Expression Analysis
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RIPA (Meilunbio, MA0151) buffer supplemented with protease inhibitors and phosphatase inhibitors was used to extract total protein from cells. The protein samples were electrophoresed on SDS-PAGE gels and transferred to PVDF membranes before being blocked with 5% skimmed milk for 1 h. The membrane was incubated with primary antibodys, including HSD11B1 (1:1500 dilution; Abbkine) and β-actin (1:50,000 dilution; Abcam) at 4°C overnight. Secondary goat anti-mouse IgG-HRP (1:5000 dilution; Abcam) antibody was incubated for 1 hour at room temperature. The proteins were visualized using ECL detection reagents (Meilunbio, MA0186) and quantitatively analyzed by Image J. Western blotting was carried out according to the experimental process of our laboratory [32 (link)].
3
Cisplatin and Raloxifene Combination Therapy
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Cisplatin (molecular formula: Cl2H6N2Pt, molecular weight: 300.05) was purchased from Jiangsu Hansoh Pharmaceutical Group Co. Ltd., and raloxifene (molecular formula: C28H27NO4S.HCl, molecular weight: 510.05) from Dalian Meilun Biotechnology Co. Ltd. RIPA (radio immunoprecipitation assay) buffer, phenylmethanesulfonyl fluoride (PMSF), BCA protein quantification kit, and the superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) detection kits, JC-1 mitochondrial membrane potential assay kit and 5-Ethynyl-2ʹ-deoxyuridine (EdU) Cell Proliferation Kit with Alexa Fluor 594 were purchased from Beyotime Biotechnology. Hematoxylin and eosin (H&E) staining solutions, cell counting kit 8 (CCK-8) were purchased from KeyGEN Biotechnology Co. Ltd. Periodic acid-Schiff (PAS) staining solutions and corn oil were purchased from Solaibao Technology Co. Ltd., ethanol and xylene from Aladdin reagents, and the creatinine and urea nitrogen detection kits from Kehua Biological Engineering Co. Ltd. Primary antibody: anti-Bcl-2, anti-Bax, anti-β-actin were purchased from Wanleibio Company (Shenyang, China). HRP-labeled secondary antibody was purchased from Proteintech Company (Wuhan, China).
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