Chemiluminescent hrp substrate detection system

Cell lysates were prepared with RIPA buffer supplemented with a protease inhibitor cocktail, and equal amounts of protein (30 μg) samples were separated in SDS-PAGE gels. For immunoblotting, the membranes were probed with primary antibodies for HDAC1 (Cat. ab19845, Abcam, Cambridge, UK), HDAC4 (Cat. Ab79521, Abcam, Cambridge, UK), and GAPDH (Cat. ARG10112, Arigo, Taiwan) overnight at 4 °C and then incubated with horseradish peroxidase HRP-conjugated goat anti-rabbit IgG (Cat. ab6721, Abcam, Cambridge, UK). Protein signals were visualized using a chemiluminescent HRP substrate detection system (BioRad, Hercules, CA, USA) and acquired by a luminescence imaging system (UVP BioSpectrum 600, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Cell lysates were extracted with RIPA supplemented with a protease inhibitor cocktail, and equal amounts of protein (30 μg) samples were separated in SDS-PAGE gels. For immunoblotting, membranes were probed with primary antibodies for Aβ (Cat. 8243, Cell signaling, Danvers, MA, USA and clone 6E10, Covance Inc, Princeton, NJ, USA), NeuN (Cat. 52283, Arigo, Taiwan), Nestin (Cat. 52345, Arigo, Taiwan), Neprilysin (Ab5458, Millipore, Bedford, MA, USA), or GAPDH (Cat. 10112, Arigo, Taiwan) overnight at 4 °C and then incubated with horseradish peroxidase (HRP)-conjugate goat anti-mouse IgG (1:10000, ab136815, Abcam, Cambridge, UK) or HRP-conjugate goat anti-rabbit IgG (1:1000, ab6721, Abcam, Cambridge, UK). Protein signals were visualized using a chemiluminescent HRP substrate detection system (BioRad, Hercules, CA, USA) and acquired by a luminescence imaging system (UVP BioSpectrum 600, Thermo Fisher Scientific Inc., Waltham, MA, USA).