Blocking reagent for can get signal

Aβ42 solution (10 μM) was incubated with or without each compound (DDC and 1–3, 10 or 50 μM) at 37 °C for 1 h. 1 μL (45 ng) of each Aβ sample was applied to the nitrocellulose membrane (0.2 μm pore size, Biorad), and the membrane was blocked with Blocking Reagent for Can Get Signal (TOYOBO, Tokyo, Japan). The membrane was then probed with A11 antibody^{39 (link)} at 1 μg mL⁻¹ (Invitrogen, Carlsbad, CA), followed by treatment with the secondary antibody (anti-rabbit IgG or anti-mouse IgG). Development was performed with ECL chemiluminescence (GE Healthcare). The first and second antibodies were diluted with Can Get Signal Solution 1 and 2 (TOYOBO), respectively. Phosphatase buffered saline plus potassium (PBS-K; 1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, 140 mM NaCl, 2.7 mM KCl, pH 7.4) including 0.5% Tween-20 was used as a washing buffer.

Cultured cells were lysed in RIPA buffer (10X RIPA buffer and protease inhibitor
cocktail, Cell Signaling Technology). Cell lysates (20-30 μg of protein/lane)
were subjected to electrophoresis on a polyacrylamide gel and then transferred
to a Trans-Blot Turbo PVDF membrane (Bio-Rad Laboratories, Hercules, CA).
Membranes were blocked with 5% skim milk or Blocking Reagent for Can Get Signal
(TOYOBO, Osaka, Japan) and incubated with primary antibody overnight at 4°C. The
membranes were incubated with second antibody for 1 hour at room temperature.
Immunoblots were visualized using ECL Prime Western Blotting Detection Reagent
(Cytiva) with an LAS-3000 (Fujifilm, Tokyo, Japan) and ImageQuant LAS 500 imager
(Cytiva).