Image reader las 4000

Cells were lysed, and protein extracts were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking the membranes, rabbit polyclonal antibodies against FOXO4 (1:1000, ab128908, Abcam, Cambridge, UK) and PHLPP2 (1:1000, ab71973, Abcam), and a GAPDH monoclonal antibody (1:4000, #2118, Cell Signaling Technology, Danvers, MA, USA) were incubated with the membranes. The anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5000, #7074, Cell Signaling Technology) were then added to the blots and the blots were exposed using an Image Reader LAS-4000 (Fujifilm, Japan).

Log phase yeast cultures were harvested through centrifugation, and total protein extracts were prepared as described previously. In short, 200 μl 1.85 M NaOH was added to 900 μl culture sample and incubated for 10 min on ice. Then 200 μl of 55% TCA was added and mixed gently by inverting the sample tubes. The suspension was incubated further for 10 min on ice. Cells were pelleted using centrifuge (13,000 g) and later resuspended in 100 μl HU buffer (8 M Urea, 5% SDS, 200 mM Tris pH 6.8, 1 mM EDTA, 1.5% [w/v] DDT, 0.1% [w/v] bromophenol blue) per 1 OD₆₀₀ equivalent of sample culture. Samples were incubated at 65°C for 10 min before analysis by SDS-PAGE.
Western blot analysis: Proteins resolved by SDS-PAGE were transferred to PVDF membranes by TransBlot Turbo semi-dry blotter from Bio-Rad. Transferred membranes were then blocked with 3% BSA (w/v) in TBS-T. Specific primary and secondary antibodies (conjugated with Alkaline Phosphatase) were used to detect proteins of interest (antibodies are listed in Table S6). Protein bands were visualized by probing the membrane with ECF reagent (GE Healthcare) and later imaging with ImageQuant LAS-5000 (Fujifilm) and the software Image-Reader LAS-4000 (Fujifilm).