Anti fsp1 antibody

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).

For in vivo studies, paraffin-embedded heart tissue sections were deparaffinized and rehydrated, blocked in 10% bovine serum albumin (BSA) (Thermo Fisher Scientific, MA, USA), and incubated with primary antibodies, specific anti-GFP antibody (1:100 dilution; ABclonal, AE011), anti-FSP1 antibody (1:50 dilution; Abcam, ab93283), anti-Snail1 antibody (1:100 dilution; eBioscience, 14-9859-82), and anti-MCT1 antibody (1:100 dilution; ABclonal, A9061) at 4°C overnight. For in vitro studies, endothelial cells were fixed with 3.7% formaldehyde (Sigma-Aldrich, MO, USA), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, MO, USA) in phosphate-buffered saline (PBS), and blocked with 3% BSA in PBS followed by incubating with primary antibodies at 4°C overnight. The following primary antibodies were used for immunofluorescent staining: anti–VE-cadherin antibody (1:50 dilution; Abcam, ab33168), anti-CD31 antibody (1:50 dilution; Abcam, ab28364), anti-FSP1 antibody (1:50 dilution; Abcam, ab93283), and anti-Snail1 antibody (1:100 dilution; eBioscience, 14-9859-82). The next day, slides or cells were stained with secondary antibodies at room temperature for 90 min and mounted in mounting medium with DAPI (Vector Laboratories, CA, USA).