Phenyl methyl sulfonyl fluoride (pmsf)

The paradigm used was like those described previously [40 (link), 47 (link)]. The hippocampal tissues of the mice were homogenized in ice-cold RIPA Lysis Buffer (Beyotime Biotech) supplemented with the PMSF (Selleck) and protease inhibitor (Millipore). The homogenates were centrifuged at 1000g for 15 min at 4 °C. Twenty μL of each sample being stored for BCA assay and the rest was mixed with 4 × SDS loading buffer (250 mmol Tris–HCl, pH 6.8, 20% β-mercaptoethanol, 4% SDS, 0.004% bromophenol blue (wt/vol), 40% (vol/vol) glycerol) in a 3:1 ratio, heated at 80 °C for 15 min. Each sample was run on an SDS-PAGE (10% acrylamide) and transferred to a PVDF membrane (Millipore). The membranes were blocked for 1 h with TBST (0.9% NaCl, 10 mM Tris, 0.1% Tween-20, PH7.4) containing 5% BSA on an orbital shaker at room temperature, and then incubated the primary antibody overnight at 4 °C (anti-amyloid precursor protein, 1:5000, A8717, Sigma-Aldrich; anti-tubulin, 1:10,000, CWbiotech). After three washes for 10 min each with TBST, the membranes were subsequently incubated with HRP-linked secondary antibody (goat × rabbit/mouse, HRP-linked, 1:10,000, KangChen Biotech Inc, China) for 2 h at room temperature. Immunoreactivity was detected by using Gel Imaging System (Tannon 5200 Multi) and analyzed by using the Image J software.

Total cell lysates were obtained using RIPA buffer (CWBIO) containing protease inhibitor PMSF (Selleck) and dithiothreitol (DTT) (Sigma). Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). The bands were visualized using the ECL chemiluminescence kit (Monad). Nuclear-cytoplasmic fractions were obtained using the Nuclear and Cytoplasmic Protein Extraction Kit (catalog no. P0027; Beyotime), then subjected to western blotting. Antibodies used are shown in Supplementary Table S2.

Western blotting assays of ATC cells were implemented at 48 h after treatment. First of all, ATC cells were lysed by RIPA comprising 1 mM PMSF (Selleck, USA) and the concentration of total proteins was quantified using the bicinchoninic acid kit (Beyotime, China). Secondly, 40 μg of proteins was separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Pall, USA). Then, membranes were blocked using 5% nonfat milk (Beyotime, China), hatched with primary antibodies overnight at 4°C, and subsequently incubated with secondary antibody (1:10,000) (Affinity, China) for 1 h at 37°C. Finally, blots were detected via ECL-PLUS kit (Beyotime, China) and analyzed using ImageJ software (version 1.8.0, NIH). The primary antibodies used in this study were bought from Abcam (Cambridge, USA) and listed as follows: anti-AKIP1 (1:500, ab135996), anti-p-PI3K (1:1,000, ab182651), anti-PI3K (1:1,500, ab86714), anti-p-AKT^Ser473 (1:1,000, ab81283), anti-AKT (1:1,500, ab8805), anti-β-catenin (1:1,000, ab68813), anti-GAPDH (1:5,000, ab181602), and anti-Histone H3 (1:5,000, ab1791).