Ab155101

Samples were lysed by the radio immunoprecipitation assay (RIPA) lysis buffer (Boster, Wuhan, Hubei, China) containing protease inhibitors at 4°C for 15 min. After 10 min of centrifugation at 4°C, the supernatants were harvested for quantification using the bicinchoninic acid method (Boster). Following this, the protein solution was subjected to 10% SDS-PAGE followed by electro-transferring onto a PVDF membrane. The membrane was blocked with 5% skim milk at indoor temperature for 0.5 h and incubated with the primary anti-WIF1 (1:2,000 dilution; #ab155101; Abcam, Cambridge, UK), MMP-2 (1:2,000 dilution; #ab92536; Abcam), MMP-9 (1:1,000 dilution; #ab76003; Abcam), Vimentin (1:2,000 dilution; #ab137321; Abcam), E-cadherin (1:500 dilution, #ab231303), DNMT1 (1:1,000 dilution; #ab188453; Abcam), DNMT3A (1:500 dilution; #ab2850; Abcam), DNMT3B (1:1,000; #ab2851; Abcam), and GAPDH (1:5,000 dilution; #ab8245; Abcam) at 4°C for 12 h. Subsequently, membrane was hatched with goat anti-rabbit (1:10,000; #ab205718; Abcam) or rabbit anti-mouse (1:5,000; #ab6789; Abcam) secondary antibody for 1 h, and proteins were developed using the enhanced chemiluminescence kit (Millipore) followed by quantification using the Image J Software (NIH, Bethesda, MD, USA).

Total proteins were extracted from VSMCs or the aorta tissues according to the manufacturer’s instructions. In brief, equal amounts of protein was separated by SDS-PAGE and transferred onto an activated PVDF membrane (Amersham Biosciences, Piscataway, NJ, USA) after electroblotting. After blocking with 5% non-fat milk for 2 h, the membrane was incubated with the primary antibodies overnight at 4 °C. The detailed information of primary antibodies were as follow: GADPH (ab181602, Abcam, 1:1000), phosphorylated-β-catenin (ab75777, Abcam, 1:500), β-catenin (ab32572, Abcam, 1:5000), Wnt inhibitory factor-1(WIF) (ab155101, Abcam, 1:2000), Runx2 (ab23981, Abcam, 1:1000), phosphorylated-AKT (ab81283, Abcam, 1:5000), Protein Kinase B (AKT) (ab188099, Abcam, 1:2000), receptor activator of NF-kB Ligand (RANKL) (ab239607, Abcam, 1:1000) and alkaline phosphatase (ALP) (ab228636, Abcam, 1:1000), phosphorylated-STAT3 (ab76315, Abcam, 1:2000), STAT3 (ab109085, Abcam, 1:1000), BMP2 (ab214821, Abcam, 1:1000). The membrane was rinsed with TBST three times (5 min/once), and then incubated at RT for 2 h with secondary antibody. After incubation, the membrane was rinsed three times with TBST (5 min/once). By using an enhanced chemiluminescence kit, protein bands were visualized by GeneGnome chemiluminescence imaging system.