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Nutrients 11:2073

Snap frozen rat and mouse heart tissues were homogenised using Percellys tubes (approximately 10 mg tissue/300 L 50 mM HEPES (pH 7.4) buffer containing 10 mM IAM-TPP two times for 30 s)

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In the average intensity maps, each pixel indicates the localization of an individual detected molecule

The membranes were blocked with 5% BSA for 90 min, followed by incubation with the corresponding primary antibodies at 4 C overnight, including mouse anti-GAPDH (Proteintech, China, 60004-1-Ig, 1:5000), rabbit anti-Col1a1 (Proteintech, China, 25870-1-AP, 1:3000), rabbit anti-TGF-1 (Selleck, China, F1624, 1:1000), mouse anti-Smad3 (Proteintech, China, 66516-1-Ig, 1:6000), rabbit anti-p-Smad3 (ABclonal, China, AP0727, 1:1000), rabbit anti-Smad7 (Proteintech, China, 25840-1-AP, 1:1000), mouse anti-PI3K (Proteintech, China, 60225-1-Ig, 1:20000), rabbit anti-p-PI3K (Affinity, China, AF3241, 1:1000), rabbit anti-AKT (ABclonal, China, A17909, 1:3000), rabbit anti-p-AKT (Cell Signaling Technology, USA, 4060S, 1:2000), and rabbit anti-Hmox1 (Proteintech, China, 10701-1-AP, 1:1000) antibodies

For long-term storage, keep it in a freezer