Ecl select substrate

Protein was extracted with RIPA buffer. 30 μg total protein was loaded onto SDS-PAGE and separated by electrophoresis. Separated proteins were then transferred to PVDF membrane followed by blocking with 5% BSA in TBST buffer (25 mM Tris-HCl, 137 mM NaCl, and 2.7 mM KCl, 0.05% Tween-20, pH 7.4 ± 0.2) for 2 h at room temperature. Membranes were then incubated with primary antibody of interests overnight at 4 °C and appropriate secondary antibody for 2 h at room temperature. The membrane was probed with ECL select substrate (GE Healthcare, Germany) on the Chemidoc chemiluminescent platform (Biorad, USA).

Protein from cell or tissue samples was extracted using RIPA buffer and loaded onto the SDS-PAGE for separation by electrophoresis. Proteins were then transferred to the PVDF membrane. Followed by blocking by 5% BSA in TBST buffer (25 mM Tris-HCl, 137 mM NaCl and 2.7 mM KCl, 0.05% Tween-20, pH 7.4 ± 0.2) for 2 hours at room temperature, the membranes were incubated with the appropriate primary antibodies of interest overnight at 4 ⁰C and secondary antibodies for 2 hours at room temperature. The membrane was read with ECL select substrate (GE Healthcare, Germany) on the Chemidoc chemiluminescent platform (Biorad, USA).

For Western blotting, samples were resolved via SDS-PAGE on 12% tris-tricine gels at a constant 45 mA per gel for 1:45 h using the Mini-PROTEAN Tetra Cell (Bio-Rad, Hercules, USA). Proteins were transferred to a PVDF membrane with a pore size of 0.2 μm (Trans-Blot Turbo Mini PVDF Transfer Pack, Bio-Rad, Hercules, USA) at 25 V, 1.3 A for 7 min, using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, USA). Membranes were boiled in PBS for 5 min in a microwave oven after transfer. After cooling down to room temperature, the membranes were incubated in fresh PBS for 5 min and in TBS-T (0.1% Tween20) for 5 min. Membranes were blocked with 2% nonfat dried milk powder in TBS-T for 1 h at room temperature. Anti-Aβ antibodies 6E10 (RRID:AB_662798, Covance, Princeton, USA), Nab228 (RRID:AB_476770, Sigma-Aldrich, St. Louis, MO, USA) or IC16 (HHU Düsseldorf, Germany) were used at a concentration of 1 μg/mL in TBS-T over night at 4 °C. Following three 10 min washes with TBS-T, the membranes were incubated with an HRP-coupled goat anti-mouse IgG (RRID:AB_228307, Thermo Fisher Scientific, Waltham, MA, USA), diluted 1:10000 in TBS-T. After washing three times for 10 min with TBS-T, protein bands were visualized with a ChemiDoc MP System (Bio-Rad, Hercules, USA) using ECL Select substrate (GE Healthcare, Boston, USA).

We extracted proteins from AML12 cells and liver tissues with RIPA lysis buffer, and the protein samples were subjected to centrifugation at 14,000 rpm at 4 °C for 15 min. After quantification, the protein lysates (20 μg) were separated on a 10% SDS–polyacrylamide gel. The separated protein was transferred to a membrane, and the protein membrane was blocked with 5% BSA in TBS-T solution for 2 h. After washing, the blocked membranes were immunoblotted with the targeted antibodies overnight at 4 °C and then incubated with HRP-conjugated secondary antibodies (1:1000). The reactive band signal was visually detected by ECL select substrate (GE Healthcare, Germany) on the Chemidoc chemiluminescent platform (Bio-Rad, USA).