Quantitative one image analysis

Equal quantities of protein (30 µg) were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked in 5% BSA at room temperature for 1 h. Then, the membranes were incubated with the following primary antibodies overnight at 4 °C: rabbit HDAC1, rabbit HDAC2, rabbit HDAC3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, 65816), rabbit IP3R (1:2000, Abcam, ab108517), rabbit p-CaMKⅡ (1:1000, Cell Signaling Technology, 12716), rabbit CaMKⅡ (1:1000, Proteintech, 13730-1-AP), rabbit ITPKA (1:1000, Proteintech, 14270-1-AP), and rabbit β-actin (1:2000, Proteintech, 20536-1-AP). The PVDF membranes were washed three times with 1× TBST and incubated with HRP-linked secondary antibody (Beyotime Biotechnology, Shanghai, China) at room temperature for 1 h. Finally, these membranes were incubated in an Amersham ECL Western Blotting Detection Kit (GE Healthcare Life Sciences, Chicago, IL, USA) for 30 s–2 min, and the signals were visualized by exposure to Quantitative One Image Analysis (Bio-Rad, Hercules, CA, USA).

The cells were lysed on ice with precooled lysis buffer. After centrifugation at 12,000 rpm for 15 min at 4°C, the total protein concentration of each experimental group was quantified using a BCA kit (Beyotime Biotechnology, China). Equivalent protein lysates (30 μg) were separated by 10% SDS-PAGE gels and blotted onto PVDF membranes (Millipore, USA). Proteins were detected by using antibodies anti-APP (1:1000, Sigma, USA) and anti-β-actin (1:1000, Santa Cruze, USA) overnight at 4°C. After incubation with the secondary antibody, signals were visualized using an enhanced chemiluminescence detection kit (Advansta, USA) and quantified using Quantitative One Image Analysis (BioRad, USA).