Image studio lite ver 4

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.

Synchronized worms of each strain were grown on OP50 plates at 20 °C and harvested at adult day 1 using M9 buffer, followed by liquid nitration freezing. Worm pellets were boiled in SDS loading buffer and loaded to replicate SDS-polyacrylamide gel electrophoresis gels. The transferred fluorescence PVDF membranes (Millipore) were probed overnight at 4 °C with anti-phospho-eIF2α (Ser51) (1:1000 dilution, Cat#3398S, Cell Signaling Technology) and anti-eIF2α (1:500 dilution, kindly provided by Dr. Shin Takagi) primary antibody, respectively. The blots were visualized by 1 h incubation at room temperature with IRDye 800CW fluorescent secondary antibodies (1:10,000 dilution, Cat#926-32211, LI-COR Biosciences), followed by scanning in the LI-COR Odyssey Infrared Imaging System according to the manufacturer’s instruction. Images were quantified with Image Studio Lite Ver 4.0 (LI-COR). The significance of intensity difference was evaluated by paired t test in Excel 2013. To examine the AKT-1::GFP level, nitrocellulose membranes were probed with anti-GFP (1:3000 dilution, Cat#11814460001, Roche) or anti-tubulin (1:5000 dilution, Cat#T3526, Sigma-Aldrich) antibodies. Horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution, Cat#AP124, Sigma-Aldrich) were used for detection.