Mouse anti yap

IMR-90 cells were cultured and then fixed by 2% formaldehyde in PBS for 15 min at room temperature before staining. 0.5% Triton X-100 was used for permeabilization and 5% BSA in PBS was used for blocking at room temperature, followed by incubation with a primary antibody including mouse anti-YAP (1:400; Proteintech), rabbit anti-p-YAP (1:400; Affinity, Melbourne, Victoria, Australia), rabbit anti-TAZ (1:400; Cell Signaling) and rabbit anti-p-TAZ (1:400; Cell Signaling) overnight at 4 °C. A fluorophore-conjugated secondary antibody against the primary antibody was used, and then the sample was covered with mounting medium containing DAPI. Samples were imaged by confocal fluorescence microscopy equipped with a camera and imaging software as the above mentioned at 200× magnification.

Protein from cells and fetal lungs was collected with lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Samples were electrophoresed in 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with non-fat dried milk diluted in Tris-buffered saline/Tween-20 (TBST) for 1 h. Samples were incubated with primary antibodies including mouse anti-YAP (1:1000; Proteintech, Rosemont, IL, USA), rabbit anti-p-YAP (1:1000; Abcam, Cambridge, UK), rabbit anti-TAZ (1:1000; Cell Signaling, Danvers, MA, USA), rabbit anti-p-TAZ (1:1000; Cell signaling), rabbit anti-FGF10 (1:1000; Abcam), rabbit anti-FGFR2 (1:1000; Abcam), rabbit anti-SOX2 (1:1000; Cell Signaling), rabbit anti-SOX9 (1:1000; Cell Signaling), rabbit anti-SIRT1 (1:1000; Cell Signaling), rabbit anti p-SIRT1 (1:1000; Signalway Antibody, Greenbelt, MD, USA), and mouse anti-β-actin (1:5000; Proteintech) overnight at 4 °C. After incubation with secondary antibodies for 1 h at room temperature, protein bands were detected with the ChemiDoc™ MP Imaging system (Bio-Rad, Hercules, CA, USA) and quantified by Image-Pro software (vers. 4, Media Cybernetics, Rockville, MD, USA). All data were normalized to the control.