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2.7 Aortic ring assay The mouse aortic ring assay was conducted according to a previously described method ( 2.8 Western blotting Proteins were extracted from the tissue samples, C2C12 myocytes, or HUVECs using RIPA lysis buffer containing protease and phosphatase inhibitor cocktails (Nacalai Tesque, Kyoto, Japan), separated using SDS-PAGE, and transferred to PVDF membranes

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Although ICH Q2(R2) is primarily intended for pharmaceutical analytical procedures, its fit-for-purpose validation principles may be used as a non-mandatory scientific reference for research-reagent testing