Phosstop easypack

Total protein lysates were collected in RIPA buffer supplemented with protease (cOmplete™ ULTRA Tablets, Mini, EDTA-free, EASYpack Protease Inhibitor Cocktail, Sigma) and phosphatase (PhosSTOP™, EASYpack, Sigma) inhibitors. Lysate were sonicated (5 cycles, 10 seconds on, 15 seconds off) using a Bioruptor® 300 (Diagenode). Protein concentrations were quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific) and 40-100μg of protein was loaded for blot analysis after heat denaturation in 1X sample buffer (60mM Tris-HCl, pH 6.8, glycerol, 2% (w/m) SDS, 0.005% β-ME). Fractionation was carried out by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) or nitrocellulose membrane (0.2 μm) by overnight wet transfer. Membranes were blocked in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) plus 5% nonfat milk for 1 hour at room temperature. Primary and secondary antibody incubations were performed overnight at 4°C and 1 hour at room temperature, respectively, at the recommended dilutions. Blots were developed according to the manufacturer’s recommendations with Clarity Max™ Western ECL Substrate (Biorad) for HRP-conjugated secondary detection, or with the Odyssey (Li-Cor Biosciences) system. ImageJ⁷⁵ (link) was used to quantify band intensities.

Whole-cell lysates were prepared using a radioimmunoprecipitation assay buffer with cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Sigma-Aldrich Japan, Tokyo, Japan) and PhosSTOP EASYpack (Sigma-Aldrich, Tokyo, Japan). Anti-FOXM1 monoclonal (1:2000; ab207298; Abcam, Cambridge, MA, USA) and anti-β-actin monoclonal (#4967; Cell Signaling Technology, Danvers, MA, USA) antibodies were used. Immune complexes were visualized using Amersham ECL Prime Western Blotting Detection Reagents (RPN2232; GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions.

Protein samples from the frontal cortex, entorhinal cortex and hippocampus were extracted from each of the stock sheep brains (n = 30) used in this study. Tissue was homogenised in 10 volumes of homogenisation buffer (TBS (50 mM Tris/HCl, pH 7.4) with 1 tablet of cOmplete™, Mini EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich^®), and 1 tablet of PhosSTOP EASYpack (Sigma-Aldrich^®) phosphate inhibitor. Homogenised tissue was centrifuged at 27,000×g for 1 h. The supernatant was collected as the soluble fraction and stored at − 80 °C until use.