2.8 Immunofluorescence cell staining
Cultured VSMCs were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton-X-100 (Beyotime, China) for 30 min. Then cells were washed three times with PBS and incubated with anti-phospho-Smad3 (anti-p-Smad3) antibody (1:200 dilutions, ab52903, Abcam, England) overnight at 4°C, followed by secondary goat anti-rabbit IgG-Cy3 antibody (1:200 dilutions, A0516, Beyotime, China) incubated for 1 h at room temperature. Nuclear staining of the cells involved using 4’6-diamidino-2-phenylindole dihydrochloride (DAPI, Beyotime, China) for 5 min at room temperature. Images were captured by fluorescence microscopy (OLUMPUS, Japan).