Anti wnt4

For isolation of cytoplasm and nuclear fractionation, HASMCs were harvested and suspended in isolation buffer A containing protease inhibitors (HiScript II First Strand cDNA Synthesis Kit; Vazyme Biotech, Nanjing, Jiangsu, China). After rotating for 1 minute and centrifuging at 12000 g for 5 minutes, supernatant with cytoplasm fraction was collected. The debris was suspended in isolation buffer B containing protease inhibitors, and then rotated for the collection of nuclear fractionation. For western blot analysis, proteins (30 µg) extracted from HASMCs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then electro-transferred onto nitrocellulose membrane. Membranes were blocked in 5% skim milk, and incubated with primary antibodies: anti-Wnt4 (1:1500; Abcam, Cambridge, MA, USA), anti-βcatenin (1:2500; Abcam, Cambridge, MA, USA), anti-Histone, anti-GAPDH and anti-β-actin (1:3000; Abcam, Cambridge, MA, USA). After incubation with horseradish peroxidase labeled secondary antibody (1:5000; Abcam, Cambridge, MA, USA), the signals were determined by Amersham enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Little Chalfont, UK).