Foxa2 antibodies

Nurr1/Foxa2 protein interaction in the VM was further analyzed by PLA. Frozen slices of mouse VM tissues (10 weeks old) were treated with primary anti-Nurr1 (1:1,000, mouse, R&D Systems) and Foxa2 antibodies (1:1,000, goat, Cell Signaling), followed by secondary antibody, ligation, polymerization, and detection using an in situ PLA assay kit (Olink Bioscience, Uppsala, Sweden), as described previously (Yi et al, 2014 (link)).

Cells were lysed in a RIPA buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 10 mm NaF, 0.1% SDS, 1% Nonidet P-40, and 1× protease inhibitor cocktail) (Pierce, IL, USA). Cell lysates were then collected by centrifugation at 12,000× g for 20 min at 4 °C. A bicinchoninic acid (BCA) assay was conducted to measure protein concentration. Equal amounts of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF, Bio-Rad) membranes. The membranes were then blocked with 5% milk for 1 h at room temperature followed by an incubation with primary antibodies in milk overnight at 4 °C. The membranes were washed with TBS-T and subsequently probed with an HRP-conjugated secondary antibody (1:5000) at room temperature for 2 h. Signals were visualized using ECL (Millipore or Bio-Rad, Canada) according to the manufacturers’ protocols. CCNG2 antibody was obtained from Abcam (1:500). β-catenin and FOXA2 antibodies were obtained from Cell Signaling and diluted to 1:1000 and 1:2000, respectively. FOXO3 (1:1000), Lamin B (1:2000), and GAPDH (1:10,000) antibodies were purchased from Santa Cruz.