Phosphor yap ser127

For Western blotting cells were lysed with RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO) containing protease inhibitor cocktail and Halt™ phosphatase inhibitor cocktail (Pierce Biotechnology), centrifuged at 14,000 × g, and supernatant was collected. Total protein concentration was determined by BCA protein assay kit (Pierce Biotechnology, Foster City, CA, USA). Samples (40 µg/each well) were SDS–PAGE electrophoresed using Criterion XT system for high molecular weight proteins (XT-running buffer, XT-sample loading dye and 3–8% Tris–acetate pre-casted gels Bio-Rad, Hercules, CA, USA), blotted onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad) and immunoreacted with antibodies to FAT1 (Abcam: ab190242), YAP/TAZ (Cell Signaling Technology: 8418), phosphor YAP- Ser127 (Cell Signaling Technology:13008), and β-ACTIN (Cell Signaling Technology: 3700) for normalization (1:1000 dilution). The membranes were then incubated with secondary antibodies (LI-COR, goat anti-mouse IRDye 800CW or donkey anti-rabbit IRDye 680RD, 1: 20,000 dilution) for 45 min at room temperature followed by washings, scanned using the Odyssey infrared scanner and analyzed using Image Studio Lite Ver.4.0 (LI-COR Inc., Lincoln, NE, USA). Uncropped scan of the blots is provided in the Supplementary Information section.