Protein Extraction and Analysis from Tissues and Cells

Protein samples were extracted from tissues and cells with the same procedures described previously52 (link)53 (link). Briefly, heart tissues and HAECs were lysed in RIPA buffer and then centrifuged at 4 °C 13500 rpm for 15 minutes. The supernatant was collected, and protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China). Protein extracts were separated by SDS–PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked and incubated with the following primary antibodies: anti-VE-cadherin antibody, anti-CD31 antibody, anti-α-SMA antibody, anti-FSP1 antibody, anti-Snail antibody (Abcam, Cambridge, MA, USA), anti-p-AKT antibody, anti-AKT antibody, anti-p-GSK-3β antibody, anti-GSK-3β antibody (Wanleibio. Shenyang, China), and anti-GAPDH antibody (Kangcheng, Shanghai, China); GAPDH was used as a loading control. After incubation at 4 °C overnight, the membrane was washed three times with PBST, incubated with fluorescence-conjugated goat anti-rabbit IgG and goat anti-mouse IgG for one hour at room temperature (1:10000, Invitrogen) and scanned by Odyssey Imaging System (LI-COR, Inc., Lincoln, NE, USA).