Ecl substrate kit

A protein extraction kit (Abcam, USA) was used to extract the proteins from SO-RB50 and HXO-RB44 cells. A bicinchoninic acid assay kit (Beyotime, China) was used to determine the protein concentration. Samples (15 μg) were subjected to constant-voltage electrophoresis at 80 V in 10% sulfate-polyacrylamide gel. Next, the protein was transferred to a polyvinylidene fluoride membrane under a constant current of 200 mA, and the target protein membrane was cut out and sealed with skimmed milk powder at 37°C for 90 min. After blocking, the protein primary antibodies (PFKFB2, cat#bs-5005 R; GAPDH, cat#bs-0755 R; dilution ratio: 1:500; Bioss, China) were incubated with membrane overnight at 4°C. The next day, the protein secondary antibodies corresponding to the primary antibodies were incubated again for 2 h. Finally, the protein bands were visualized using enhanced chemiluminescence (ECL) substrate kit (Amersham Biosciences, Little Chalfont, UK) and analyzed using Image Lab Software (version 4.1; Bio-Rad) [37 (link)].

Treated HepG2 and SMMC-7721 cells were lysed using lysis buffer containing a protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO, USA). Total protein concentrations were analyzed using a BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Heat-denatured proteins (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis based on molecular weight using 8% gels and subsequently transferred to polyvinylidene difluoride membranes (Millipore). The transferred proteins were then blocked using 5% skim milk (BD Biosciences) for 2 h at room temperature and then incubated with primary antibodies (anti-β-catenin and anti-(p)-β-catenin) at the appropriate dilution at 4 °C overnight. The next day, membranes were incubated with the secondary antibody. Signals were detected using an enhanced chemiluminescence (ECL) substrate kit (Amersham Biosciences, Inc., Piscataway, NJ, USA) and detection system (Amersham Biosciences). Mouse anti-β-catenin (1:1000; BD Transduction Laboratories, San Jose, CA, USA) and mouse anti-(p)-β-catenin antibodies (1:1000; Cambridge, MA, USA) were used as the primary antibodies. Mouse anti-GAPDH monoclonal antibodies (1:5000; Cell Signaling Technology, Beverly, MA, USA) were used as an internal control.

Spinal cord, hippocampus, and cortex samples from both RIPA and sarkosyl extractions were resolved by SDS-PAGE or dot blot. Blots were probed with tau antibodies [TAU5, (1:1000), AT8 (1:1000), AT100 (1:1000) and AT180 (1:250)], and autophagy-related antibodies (anti-LC3, 1:500, Novus Biologicals; anti-p62, 1:1000, BD Biosciences). After overnight incubation at 4°C, blots were incubated in horseradish peroxidase-labeled secondary antibodies and visualized with an ECL substrate kit (Amersham). Band densities were analyzed with NIH Image J software and band values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Dot blots were prepared by pipetting 1.2 ul of each sample in each square of a nitrocellulose membrane and allowed to dry for 30 minutes. Blots were incubated with T22 (1:250), overnight at 4°C followed by incubation with a horseradish peroxidase-labeled secondary and visualization with ECL. Dot intensities were analyzed with NIH Image J software.